FIFTH CANADIAN SYMPOSIUM ON TELOMERES AND TELOMERASE

UNIVERSITY OF CALGARY CALGARY, ALBERTA

MAY 11-14, 2006

WELCOME TO THE

FIFTH CANADIAN SYMPOSIUM ON TELOMERES AND TELOMERASES

Calgary, Alberta May 11th – 14th, 2006

We are excited and very pleased to welcome you to the fifth Canadian

Telomere Symposium. Telomeres remain a “hot” topic and we hope this gathering will allow, as in previous meetings, a fruitful exchange of information and expertise in an informal setting. Thanks for all the prompt E-mail responses and suggestions that we received during the organization of the meeting and we hope you can enjoy your stay in Calgary.

This is the final mailing before the meeting and you should find in these few pages all pertinent information on the workshop:

• Financial aspects • How to find University of Calgary • Program

Should there be anything else you need to know before the meeting and

that is not covered here, drop an e-mail, and we will make every effort to help or put you in touch with someone who can.

The organizers

Tara Beattie, Karl Riabowol, Raymund Wellinger, Chantal Autexier, Lea Harrington and Peter Lansdorp

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Finances Grateful thanks to all the sponsors who made our symposium possible: Major Sponsors:

◊ The National Cancer Institute of Canada ◊ The Alberta Cancer Board

◊ The Canadian Institutes of Health Research

Through the CIHR Institute of Aging

◊ The Alberta Heritage Foundation for Medical Research

Alberta Heritage Foundation for Medical Research

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Upon Arrival in Calgary ossible) in taxis to get from the airport to the

ccomodations and Lecture Hall

Please car-pool (where pUniversity. The cost for a one way fare is approximately $35. A

sing will be housed in Cascade Hall

ay 11. Check out is by

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All conference participants requiring houat the University of Calgary (see attached map, denoted by blue arrow). You will receive your room assignments upon check-in. Accommodation check-in begins at noon Thursday M11:00 am on Sunday May 14, 2006. F

f the meals will be provided at the Terrace Lounge, which is adjacent

resentations

Most oto the lecture halls. P

ations will take place in Cassio A/B which is located at on will

map of the campus and directions how to find your way from the residence

mergency Phone Contacts

All oral presentMacEwan Hall at the University of Calgary (see map). The poster sessibe held in the Escalus Room. Ato the conference is appended. E

03-220-8328

ymposium Program

Tara Beattie Office: 4Karl Riabowol Office: 403-220-8695 Tiz Reiter Office: 403-220-3029 S

the symposium with the presentation of our key-note

hris Counter is currently Associate Professor of Pharmacology and de

a

At 7:30 we will beginspeaker, Dr. Christopher Counter. CRadiation Oncology at the Duke University Medical Center. He has maseveral seminal discoveries. In the early 1990’s, in collaboration with SilviBacchetti and Calvin Harley, he was the first to demonstrate the relevance of telomerase and telomere stability in the development and progression of tumors, specifically in metastatic cells of the ovarian carcinoma. In 1997, as

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a postdoctoral fellow in the laboratory of Robert Weinberg, he co-discovered the telomerase reverse transcriptase TERT component in yeast. In 1999 he published, along with coauthors William Hahn and Robert Weinberg, the first creation of human tumor cells with defined genetic elements, including the human telomerase enzyme. His laboratory's research is currently focused on understanding the molecular mechanisms underlying the evolution of normal cells into cancers, specifically by studying immortalization, proliferation and creating new animal models for cancer. He has made important contributions to the field of telomerase, telomeres and cancer, publishing in high impact journals such as Nature, Cell, Proceedings of the National Academy of Sciences, Nature Genetics, Genes and Development, EMBO Reports, Cancer Cell, Current Biology, and Cancer Research. After the plenary lecture, the bar will be open for informal meetings and socializing. The symposium will reconvene on Friday morning for the first regular session (see below for session schedule). All talks will take place in the Cassio A/B rooms of MacEwan Hall at the University of Calgary. In the lecture hall, slide-projectors and computer aided presentation set-ups will be available. All speakers will have 15 minutes for their presentation followed by 5 minutes for questions and discussion. If presenting using a computer, please send your presentation to Tara Beattie at tbeattie@ucalgary.ca before the meeting or bring a copy of your presentation on a memory stick or CD. Posters: There will be 2 poster sessions on each of Friday and Saturday afternoons. Posters can be put up on Thursday and left until Saturday evening in the Escalus room of MacEwan Hall. These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with consent of the author. There will be a competition for best presentation (poster and oral, for trainees only) and the winners will be announced during the banquet on Saturday night.

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Meeting Schedule

Thursday May 11, 2006 12:00 Arrival in Calgary and Accommodation Check-In Begins 5:00-7:00 Registration 5:00-7:00 Wine and Cheese Reception – Shirley Anastasia Robertson

Lounge 7:30-7:45 Meeting Welcome and Opening Remarks – Tara Beattie and

Raymund Wellinger 7:45 Introduction – Lea Harrington 7:45-8:45 Plenary Talk by Dr. Chris Counter – Telomerase: The good, the

bad and the ugly Friday May 12, 2006 (Underlined is the presenter) 8:00-9:00 Breakfast (Terrace Lounge) 9:00-9:15 Opening Remarks Telomere Erosion and Nuclear Dynamics - Chair George Chaconas 9:15-9:35 M. Hills and P. Lansdorp– The role of oxidative Stress on normal

and atypical telomere erosion rates 9:35-9:55 M. Meznikova, N. Erdmann and L. Harrington – Tert and Stem

Cell Maintenance 9:55-10:15 K. Lisaingo, M. Schertizer and P. Lansdorp – Telomere

organization and chromosome instability in mouse embryonic stem cells

10:15-10:45 Break (Terrace Lounge) 10:45-11:05 B. Unryn, L. Cook and K. Riabowol - Telomere Length of

Offspring is positively linked to Paternal Age 11:05-11:25 J. Famulski, L. Vos and G. Chan – Kinetochore mitotic checkpoint

protein dynamics revealed by live cell fluorescence microscopy

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11:25-11:40 M. J. Hendzel, D. McDonald and C. Andrin – Nuclear Actin-Polymerization and involvement in DSB repair

12:00 – 1:00 Lunch (Terrace Lounge) 1:00-2:30 Poster Session #1 – Even numbered Posters – Escalus Room Telomeres and Telomerase in Disease States – Chair Karl Riabowol 2:30-2:50 B. Vukovic, B. Beheshti, P. Park, G. Lim, J. Bayani, M. Zielenska,

and J.A. Squire – Breakage-Fusion-Bridge as a driving force for in vitro genomic instability and karyotypic evolution in prostrate cancer

2:50-3:10 A.M Joshua, B. Vukovic, I. Braude, A. Evans, S. Hussein, J. Srigley, and J.A. Squire – Quantitative Fluorescence in-situ hybridization of telomere length in high-grade prostatic intraepithelial neoplasia predicts outcome to prostatic carcinoma

3:10-3:30 M. Taboski and L. Harrington – The role of telomerase in tumourigenesis

3:30-3:50 F. Rashid and S. Done – PPARγ Signaling and telomerase regulation in Breast Cancer

3:50-4:15 Break (Terrace Lounge) Chair Peter Lansdorp 4:15-4:35 H. Root, D. Stavropoulos and S. Meyn – Fanconi Anemia proteins

and BLM, the Bloom syndrome DNA helicase, are involved in ALT telomere length maintenance

4:35-4:55 B. M. Unryn, L. J. Bestilny, K. Riabowol and M. J. Gill - HAART blocks the accelerated telomere erosion in HIV patients

4:55-5:15 B. M. Unryn, D. Hao, S. Gluck, L. Cook and K. Riabowol – Increased acceleration of telomere loss by chemotherapy in patients with locally advanced head and neck cancer

5:30 – 7:30 Dinner (Terrace Lounge)

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Telomere and Telomerase Structure and Function – Chair Chantal Autexier 8:00-8:20 J. Fakhoury, D.T. Marie-Egyptienne, and C. Autexier – Role of

telomerase reverse transcriptase in the divergent regulation of mouse and human telomerases and telomere function

8:20-8:40 S. Larose, N. Laterrereur, S. Abou Elela and R. Wellinger – Regulation of telomerase by Rnt1p

8:40-9:00 M. Downey – The role of KEOPS complex in telomere function 9:00-9:20 S. F. Louis, B. Vermolen, Y. Garini and S. Mai - c-myc alters the

three-dimensional order of telomeres and chromosomes in the interphase nucleus

Saturday May 13, 2006 8:00-9:00 Breakfast (Terrace Lounge) Techniques in Telomere Biology – Chair Raymund Wellinger 9:00-9:20 S. D. Perrault, W.A. King and D. Betts – Real-Time PCR

assessments of telomere length, telomerase activity and gene expression response to ectopic telomerase

9:20-9:40 A. Bah, E. Gilson and R. Wellinger – Humanized yeast as a tool to study telomere biology

9:40-10:00 N. Lévesque, A. Dandjinou and R. Wellinger – An attempt to purify a telomerase holoenzyme from S. Cerevisiae

10:00-10:30 Break (Terrace Lounge) Yeast Telomerase – Chair Lea Harrington 10:30-10:50 C. Le Bel, E. Rosonina, L. Maringele, D. Lydall and L. Harrington –

Searching for genes and pathways involved in a RAD52-, telomerase-independent mechanism of survival in yeast

10:50-11:10 M. Larrivée, V. Karpov and R. Wellinger – Telomerase- and Cap-independent survivors in yeast.

11:10-11:30 C. Olivier, F. Gallardo, R.J. Wellinger, and P. Chartrand – Following the intracellular processing and localization of yeast telomerase RNA

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11:45-1:00 Lunch (Terrace Lounge) 1:00-2:30 Poster Session #2 - Odd Numbered Posters – Escalus Room DNA damage and repair and telomere length maintenance – Chair Tara

Beattie 2:30-2:50 M. Glover – BRCT domains – conserved phosphor-peptide

recognition modules in the DNA damage response 2:50-3:10 E. O’Dor and P. Lansdorp - Telomeric chromatin in

Differentiation and DNA damage 3:10-3:30 Y. Yu, A. Goodarzi, R. Ye, P. Douglas, N. Morrice, P. Jeggo and S.

Lees-Miller – Autophosphorylation of DNA-PKcs controls the endonuclease activity of Artemis by regulating access to DNA ends

3:30-4:00 Break (Terrace Lounge) Chair – Susan Lees-Miller 4:00-4:20 P. Bradshaw, D. Stavropoulos, M. Trottier, D. Gilley and S. Meyn

– Human TRF2, TRF1 and TIN2 are involved in an early DNA damage response

4:20-4:40 N. Ting, B. Pohorelic, Y. Yu, S. Lees-Miller and T. Beattie - DNA-PK and telomere maintenance: What’s new on this end?

4:40-5:00 Y. We, S. Xiao and X. Zhu – Rad 50 Antagonizes the action of TRF1 on human telomere length control

6:00 – Banquet and Closing Summary by Tara Beattie and Raymund Wellinger

(The Den)

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Map from Airport to the University

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Campus Map

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Abstracts

Oral Presentations

Telomere Erosion and Nuclear Dynamics

Chair: George Chaconas

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THE ROLE OF OXIDATIVE STRESS ON NORMAL AND ATYPICAL TELOMERE EROSION RA Mark Hills

TES

and Peter Lansdor (Terry Fox Laboratory, British Columbia Cancer Agency)

Oxidative stress has long been thought to play an important role in cellular aging. Damage from oxygen radicals to genom and telomeric DNA could limit the proliferative potential of cells in vivo and in vitro. Since telomere erosion is thought to limit the number of divisions a cell can ke and critically short telomeres lead to replicative senescence, it seems of interest to udy if the rate of telomere attrition in cells varies with the level of oxygen radicals in cells and tissue culture medium. It is possible that oxidative d lomere length, leading to an incr t undergo for a culture to reach causes atypical telomere shortening resulting in a low num er of ultra-short telomeres, but no global increase in the rate of telomere erosion.

In order to address whether some telomeres are abruptly shortened, we used s at dest the

senescence and calculate outlying telomeres. Cells grown in stressed vs. normal c nce in growth rate or cell death. Preliminary data sugges are more prevalent in cultures subjected to oxidative stress, and in one c eres may have been healed via a novel mechanism of telomere maintenance.

p

ic

ma st

amage activates apoptotic pathways independent of teease in the number of cell divisions unaffected cells musconfluence. It is equally possible that oxidative stress

b

STELA (single telomere length amplification) to analyse individual telomere lengththe Xp/Yp chromosome end. Using STELA it is possible to identify even very modifferences in average telomere lengths, and also outlying telomeres far shorter thanaverage length. We have taken BJ fibroblasts through successive population doublings to

d average telomere length andonditions showed no differets that ultra-short elomerest

ase, telom

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TERT AND STEM CELL MAINTENANCE

Marie Mezníková*, Natalie Erdmann* and Lea Harrington*

*The Campbell Family Institute for Breast Cancer Research, Ontario Cancer Institute, to, ON, M5G 2C1, Canada)

by autosomal-dominant dyskeratosis congenita, which i

oing studies will be presented. REFERENCES: Erdmann, N. et al. (2004) Proc Natl Acad Sci USA 101(16): 6080-5 Hao L.Y. et al. (2005) Cell 123(6):1121-31

(University Health Network, 620 University Avenue, Toron

We previously described the dosage sensitive telomere length maintenance by murine Telomerase reverse transcriptase (mTert). Despite haploinsufficiency for the maintenance of long telomeres, mTert +/- mice retain short telomeres at all chromosome ends and thus more closely simulate the average telomere length in humans (Erdmann et al., 2004). In addition, a murine model that retains telomerase function in the presence of short telomeres will enable a genetic test of the hypothesis that a partial loss of telomere function may predispose to age-associated diseases and cancer. For example, Hao and colleagues have recently shown that heterozygosity of mTerc predisposes to loss of hematopoietic stem cell function in a murine background with short average telomere lengths (Hao et al., 2005). These results mimic the bone marrow failure and genetic anticipation observed in patients affected

s associated with heterozygous mutations in either the RNA subunit of telomerase (hTR) or TERT. We have continued to breed mTert+/- mice with short telomeres to wild-type C57Bl/6 mice, for up to 12 generations, in order to examine further telomere attrition and/or loss of stem cell function. In addition, mTert +/- mice were crossed together, termed G10 or G12 mTert+/- ‘intercrosses’. Littermates of all three possible mTert genotypes (+/+, +/- and -/-) from such intercrosses were analyzed for telomere length, and stem cell function in bone marrow, testes and small intestine. Bone marrow derived from mTert littermates was also assessed for the ability to reconstitute the hematopoietic lineage in irradiated recipient animals. The results from these ong

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TELOMERE ORGANIZATION AND CHROMOSOME INSTABILITY IN MOUSE EMBRYONIC STEM CELLS

Kathleen Lisaingo

, Mike Schertzer, Peter Lansdorp.

Telome

cs and telomere dysfunction throughout the cell cycle and the

(Terry Fox Laboratory, BC Cancer Research Centre, University of British Columbia)

res exhibit specific spatial arrangements in a cell-cycle dependent manner. High

resolution live cell fluorescence imaging was used to study the 3-dimensional organization of telomeres over two consecutive rounds of division in mouse embryonic stem cells expressing cherry-TRF1 and histone 2B-venus fusion genes. The positioning and clustering of telomeres was further studied using 3D image analysis and FACS. The number of detectable fluorescence signals can be resolved into 63 ± 7 foci per diploid nucleus in G1 and S phase and 107 ± 16 foci in G2/M. This suggests that some telomeres are clustered in interphase cells and do not separate until the G2/M transition. The spatial organization of telomeres is important for studies of cells with known telomere dysfunction. Rtel -/- ES cells exhibit an S-phase arrest and genetic instability upon differentiation. Furthermore, undifferentiated Rtel -/- ES cells have shorter telomeres (~ 15 kb) than wild type cells. These knockout cells are therefore of interest in studying telomere dynamicontribution of telomeres to genetic instability. Live cell imaging and 3D image analysis were combined to measure the frequency of anaphase bridges and apoptosis in undifferentiated Rtel -/- mouse embryonic stem cells expressing a histone 2B-venus fusion gene.

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TELOMERE LENGTH OF OFFSPRING IS POSITIVELY LINKED TO PATERNAL AGE

rad Unryn, Linda Cook B and Karl Riabowol

(Riabowol Lab, University of Calgary)

egree of variability of telomere length seen in the human population, particularly if effects are cumulative through generations.

The ends of linear chromosomes shorten with each successive cell division primarily due to the end replication problem. Shortening occurs in vitro and in vivo in all replicating cell types except those that contain telomerase activity, such as germ cells. Loss of telomeric DNA limits the replicative lifespan of cells and forced expression of the catalytic subunit of telomerase maintains telomere length and extends the lifespan of cells in culture. Despite convincing data from studies of twins that telomere length is heritable, uniform in various tissues of individuals during development until birth, and variable from individual to individual, little is known regarding the factors that contribute to telomere length variability. Since sperm cells are unique in showing increasing telomere length with age, we asked whether the age of the father at birth would affect telomere length in their offspring. Here we show, using a random sample of 125 individuals and a blind experimental protocol, that telomere length in offspring is positively associated with paternal age, and paternal age was calculated to affect telomere length by up to 20% of average telomere length per generation. Based upon the magnitude of this effect, paternal inheritance and the vertical transmission of telomere length could account for the wide d

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KINETOCHORE MITOTIC CHECKPOINT PROTEIN DYNAMICS REVEALED

kub Famulski

BY LIVE CELL FLUORESCENCE MICROSCOPY. Ja , Larissa Vos, and Gordon Chan

epartment of Oncology, University of Alberta, Edmonton, Alberta, Canada)

The human protein Zeste White 10 is an essential component of the mitotic

t hZW10 vacates the kinetochore along MTs and toward the spindle poles in response to tension generated across sister kinetochores of aligned chromosomes. By combining structure and function studies and live cell fluorescence microscopy, we have also determined that the regulation of hZW10 kinetochore dynamics is controlled by its interaction with the hZwint-1 protein.

(D checkpoint, a failsafe mechanism for accurate chromosome segregation. hZW10 localizes to the kinetochore from prometaphase to metaphase and is responsible for the kinetochore recruitment of the microtubule (MT) motor dynein. The interaction of hZW10 with dynein has been implicated in the transport of hZW10 off the kinetochore and along MTs after chromosome alignment. The transport of hZW10 off the kinetochore is a proposed mechanism of mitotic checkpoint silencing. To determine the dynamics of hZW10 in vivo, we have generated a HeLa cell line stably expressing EGFP-hZW10 and performed Fluorescence Recovery After Photobleaching (FRAP) experiments as well as time-lapse microscopy. Our FRAP results indicate that EGFP-hZW10 rapidly turns over only at kinetochores which have achieved bi-polar attachment and are under tension. We also observed a rapid exchange of EGFP-hZW10 at the spindle poles during late prometaphase and metaphase. Interestingly, EGFP-hZW10 spindle pole turnover is also dependent on tension across kinetochores. Analysis of the time-lapse movies showed that EGFP-hZW10 first accumulates at kinetochores during prometaphase, streams off the kinetochores along MTs and toward the spindle poles during metaphase, and finally disappears from both kinetochores and spindle poles at anaphase onset. We therefore conclude tha

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NUCLEAR ACTIN-POLYMERIZATION AND INVOLVEMENT IN DSB REPAIR Michael J Hendzel, Darin McDonald, and Christi Andrin

ross Cancer Institute, 11560 University Avenue, Edmonton, Alberta, Canada T6G 1Z2)

(Department of Oncology, Faculty of Medicine, University of Alberta C It is only recently that accumulated evidence has led to the general acceptance that actin is present within and has functions specific to the nucleus. Using fluorescently-tagged β-actin and fluorescently-tagged actin binding proteins, we have studied the kinetics of actin mobility in the living nucleoplasm. Fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS) were used to assess whether the nuclear actin pool is entirely monomeric or present in small complexes, undergoes binding events that reflect functional interactions within the nucleoplasm, and/or polymerizes within the living nucleus. We find evidence for both the binding of actin generally throughout the nucleoplasm and the polymerization of actin inside the living nucleus. Approximately 20% of the nuclear actin pool recovered with similar kinetics to the polymeric fraction within the cytoplasm, was lost when cells were treated with latrunculin, which inhibits actin polymerization, and was not found for an actin mutant (R62D) which is incapable of incorporating into polymers. Based upon the identification of a kinetic signature for actin polymerization within the living nucleoplasm, we have begun to characterize potential functions for polymeric actin within the nucleus. Ongoing experiments that support a role for polymeric nuclear actin in the repair of double strand breaks within the DNA will be presented as an example of a nuclear process with an apparent requirement for actin polymerization.

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elomeres and Telomerase in TDisease States

Chairs: Karl Riabowol and Peter Lansdorp

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BREAKAGE-FUSION-BRIDGE AS A DRIVING FORCE FOR IN VITRO GENOMIC INSTABILITY AND KARYOTYPIC EVOLUTION IN PROSTATE CANCER B. Vukovic1,2, B. Beheshti2, P. Park2, G.Lim2 , J. Bayani2, M. Zielenska, and J. A. Squire1,2

(Squire Lab, Ontario Cancer Institute, University of Toronto) Chromosomal instability (CIN) is thought to underlie generation of chromosomal

sion-itotic ere is

little detailed inf N in generating genomic diversity in pros ular cytogenetic methods and array compa 45, PC3, LNCaP, 1532T

d 1542T to investigate the in vitro role of BFB as a CIN mechanism of karyotype olution. Analysis of mitotic structures in all five prostate cancer cell lines showed reased frequency of anaphase bridges of and nuclear strings in comparison to normal

ostatic stromal cell cultures. Structurally rearranged dicentrics were observed in all of investigated cell lines. Comparison to SKY data showed their integration to stable nal rearrangements. mBAND and aCGH analysis of some of the more complex

romosomal rearrangements and associated amplicons identified characteristic inverted plication, most frequently involving chromosome 8. Chromosomal breakpoint analysis owed there was a higher frequency of rearrangement at centromeric and

pericentromeric genomic regions. The mo complex rearrangements were studied by mBAND painting. The distribution of ladder-like amplifications with evenly spaced high-level amplified regions was mapped by osome14 paint pattern, and also

n , h

frequency of chromosome segments fusing with multiple recipient chromosomes, and these aberrations appeared to arise as a result of end-to-end chromosomal fusion. Telomere free end analysis indicated loss of DNA sequence. Moreover the cell lines with the shortest telomeres had the most complex karyotypes, suggesting that the reduced telomere length could be driving the observed BFB events and elevated levels of CIN in these lines.

changes and genomic heterogeneity during prostatic tumorigenesis. Breakage-fubridge (BFB) cycle is one of the CIN mechanisms with characteristic mabnormalities and the occurrence of specific classes of genomic rearrangements. Th

ormation concerning the role of BFB and CItate cancer. In this study we have used molecrative genomic hybridization analysis of DU1

anevincprtheclochdush

st

chromcharacterized at high resolution using aCGH. Adjacent spacing of focal amplificatioand microdeletions was observed, and focal amplification of end sequences were seenparticularly in the most unstable line DU145. SKY analysis of this line identified a hig

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QUANTITATIVE FLUORESCENCE IN-SITU HYBRIDISATION OF IAL

shua, A.M.

TELOMERE LENGTH IN HIGH-GRADE PROSTATIC INTRAEPITHELNEOPLASIA PREDICTS OUTCOME TO PROSTATIC CARCINOMA Jo (1,2), Vukovic, B.(1,2), Braude, I.(2), Evans, A.(3), Hussein, S (4), Srigley,

ent of Medical Biophysics, University of Toronto; 2. Division of Applied ory); 3. Department of

athology, Princess Margaret Hospital; 4. Department of Pathology and Molecular

e data suggests that both stroma and epithelial tissue are exposed to exogenous factors that predispose to neoplasia which may be manifest through telomere dysfunction.

J.(4), Squire, J.S(1,2). (1. DepartmMolecular Oncology, Ontario Cancer Institute (Squire LaboratPMedicine, McMaster University, Hamilton, Ontario) Introduction: One of the major types of genetic instability in human malignancies is characterized by structural and numerical chromosome abnormalities and is termed chromosomal instability (CIN). CIN has been demonstrated in both carcinoma of the prostate (CaP) and high-grade prostatic intraepithelial neoplasia (HPIN). This laboratory previously found a significant decrease in telomere length in both HPIN and CaP in comparison to normal prostatic epithelium and more erosion evident in HPIN in the discrete regions of the prostate containing CaP. Therefore we hypothesise that the loss of telomere integrity and chromosomal instability (CIN) is an early fundamental event in CaP oncogenesis and telomere length in HPIN associated with telomere dysfunction will provide prognostic information about progression to CaP and clinical outcome. Materials and Methods: We have analysed sextant core biopsies from 68 patients with HPIN from 1998-2000 with a median follow-up from first to last biopsy of 14.5 months. An adjacent H&E section has been obtained for each biopsy studied. To examine telomere length distribution within HPIN in the cohort, we have used quantitative fluorescence in-situ hybridisation (Q-FISH) analysis of interphase nuclei in paraffin embedded sections with directly labelled telomere specific PNA probes as the resulting fluorescence emission is known to be directly proportional to telomere length. Internal control probes for centromere normalisation intensity have also been used. Results: Statistical associations between telomere length in the HPIN sample, gene detection and the subsequent diagnosis of cancer and the Gleason score have been analysed. Logistic regression analysis suggests that telomere length in HPIN (p=0.025) and in stroma (p=0.028) predict prostate cancer. Further analyses using centromere specific probes are in progress to quantitate chromosomal instability Conclusions: These analyses reveal mechanisms underlying genomic instability in this neoplasm. Th

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THE ROLE OF TELOMERASE IN TUMORIGENESIS Michael Taboski and Lea Harrington. (Harrington Lab, Campbell Family Institute for Breast Cancer Research, Ontario Cancer Institute, University of Toronto)

NA rten

matic y the ribonucleoprotein telomerase,

hich minimally consists of a protein catalytic subunit (hTERT) and a RNA component d at

eres. eby

lifespan. The

n of

that the in

maintenance. To more precisely ascertain the effects of hTERT on the l

In this omere-

ility. g e

RAS to the sted.

clonal T

layed

Human telomeres are DNA-protein structures consisting of repetitive G-rich D

(TTAGGG) that extends 4-15 kilobase pairs at the chromosome end. Telomeres showith each cell division and limit the replicative potential of most normal human socells in culture. Telomeric DNA can be replenished bw(hTR) that contains a template for telomeric repeat synthesis. Telomerase is expresseinsufficient levels in most somatic cells to prevent critically shortened telomHowever, in 90% of human tumors telomerase is expressed at higher levels, therstabilizing telomere lengths and overcoming a limited replicative acquisition of limitless replicative potential is one of several important steps duringtumorigenesis.

Human TERT can immortalize cells through telomere maintenance andcontribute to the in vitro transformation of normal cells. Conversely, the inhibitiotelomerase in human tumor cell lines results in apoptosis. Recent studies suggestrole of hTERT in the proliferation of tumorigenic cells may be separable from its roletelomere maintenance of a tumorigenic phenotype, we are creating human tumor cells from normahuman embryonic kidney cells and human mammary epithelial cells, through expressionof the SV40 early region, H-RASV12 and a Cre-mediated excisable hTERT. manner, we can completely remove hTERT at any point, and examine the tellength dependent (or independent) effects upon tumorigenic potential and cell viabTo date, we have successfully integrated Cre-excisable hTERT in a cell line containinthe SV40 early region (HA-5 cells). These cells are not tumorigenic, as determined by thsoft-agar assay for anchorage-independent growth. We then introduced H- V12

telomerase-positive HA-5 cell line, and their tumorigenic potential is now being teUpon expression of Cre recombinase, the excision of hTERT will be confirmed incell populations. Finally, we should be able to determine unequivocally whether hTERloss leads to apoptosis and/or loss of tumorigenic potential immediately, or in a demanner consistent with the onset of critically shortened telomeres.

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PPARγ SIGNALING AND TELOMERASE REGULATION IN BREAST CANCER Fariborz Rashid, and Susan Done. (Done lab, University of Toronto)

Breast cancer is the most common malignancy afflicting North American women. In over

90% of

31 cells were treated with different concentrations of troglitazone, a ligand for PPARγ,

breast carcinomas telomerase is active. Inhibition of telomerase activity by promoting the differentiation of cancer cells could be used to treat breast cancer. Several studies suggest an important role for peroxisome proliferator-activated receptor γ (PPARγ) in induction of terminal differentiation as a potential therapeutic approach to certain human malignancies. Upon activation with its ligands, PPARγ heterodimerizes with the retinoid X receptor (RXR) and binds to a recognition sequence, DR-1, in the promoter regions of their target genes. Interestingly, RXR activation together with retinoic acid receptor (RAR) inhibits telomerase activity and cell growth in breast cancer cell lines. Given its involvement in tumorigenesis, we hypothesize that PPARγ is a potential candidate for the regulation of hTERT transcription in breast cancer.

To find a model for our study, different breast cancer cell lines were tested for the expression of PPARγ and hTERT. We found that MDA 231 is expressing both PPARγ and hTERT. MDA 2

for different time points. For each time point, total cell count, cell cycle flow cytometry, and cell viability were conducted to find the best time point and treatment dose for further experiments. Using this model, our results showed that 20µM troglitazone could arrest the cell cycle at S phase in 24 hours. Using this time point, we showed that 20µM troglitazone could reduce the expression of hTERT significantly in treated cells compared to controls. It has been shown that troglitazone arrests cell growth through the activation of p53. To test if hTERT reduction is because of p53 activation, we looked at the phosphorylation level of p53 in control and treated MDA231 cells. Our primary results showed that there are no differences between these two groups. Finally, searching in the promoter region of hTERT, we found a potential DR-1 site for the PPARγ-RXR heterodimer. In conclusion, we found that there is a cross talk between PPARγ and hTERT expression. Our primary results showed that the reduction of hTERT expression is in collaboration with MDA 231 cell cycle arrest, which is independent from p53 pathway. We are currently investigating the mechanisms by which PPARγ regulates the expression of telomerase.

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FANCONI ANEMIA PROTEINS AND BLM, THE BLOOM SYNDROME DNA HELICASE, ARE INVOLVED IN ALT TELOMERE MAINTENANCE.

eather Root H , Dmitri Stavropoulos and Stephen Meyn.

(Hospital For Sick Children and University of Toronto)

Our results indicate that the presence of FA, BLM and other recombination proteins at ALT telomeres is dynamic, with FA and BLM proteins spatially and temporally poised to assist in replication and maintenance of telomeres. Our data suggest a critical role for FANCD2 in ALT telomere maintenance and support a model in which a normal function of the FA and BLM proteins helps cells deal with dysfunctional telomeres.

Fanconi anemia (FA) patients have unusually short telomeres and BLM, the Bloom syndrome DNA helicase, unwinds telomeric DNA sequences in vitro, suggesting that these proteins may play a role in telomere maintenance. In testing this hypothesis, we have obtained evidence implicating FA and BLM proteins in ALT telomere maintenance. We find that the FA proteins FANCD2, BRCA2 (FANCD1), FANCA and FANCG localize to telomeric foci in ALT human fibroblasts, but not in telomerase-positive or primary cells. Colocalization of FANCD2 and telomeres primarily occurs within PML bodies in late S/G2. Association of FANCD2 with ALT telomeres may depend on BLM, as ~90% of FANDCD2 foci that colocalize with TRF1 also co-localize with BLM. γ−H2AX localizes to some ALT telomeric foci, suggesting that ALT cells can harbor dysfunctional telomeres that trigger a DNA damage response. FANCD2 is almost always present in those foci. BRCA2 localization to ALT telomeres occurs primarily in the presence of FANCD2, consistent with a role for FANCD2 in BRCA2 damage-induced foci formation. Co-IP experiments indicate ALT-specific in vivo interactions between FANCD2, BLM, and TRF2 in late S/G2. In support of a functional role for FA and BLM proteins in ALT, we can stably suppress FANCD2 expression with shRNA in telomerase positive cells but not in ALT cells. Additionally, reductions in FANCD2 expression negatively affects the growth of ALT fibroblasts, while reductions in BLM expression cause ALT-specific increases in dicentric chromosomes and telomeric fusions.

23

HAART BLOCKS THE ACCELERATED TELOMERE EROSION IN HIV PATIENTS Brad M. Unryn, Les J. Bestilny, Karl T. Riabowol and M. John Gill

p/year

tial. Preservation of telomere length should be considered as a potential

(Riabowol Lab, University of Calgary) HIV infection induces rapid lymphocyte turnover and the accelerated loss of telomere sequence that is believed to contribute to AIDS associated immunosenescence. In this study we asked if HAART, by reducing HIV viral load and restoring T cell counts, also arrested accelerated telomere erosion and if telomere length was restored in T cells repopulating the circulation. Blood samples from 16 HIV patients that were previously characterized for telomere length in 1995 and subsequently treated with HAART, were collected and telomere lengths of peripheral blood mononuclear cells (PBMCs) were determined and compared in parallel, pre- and post-HAART. Telomere length was measured in the same gels by terminal restriction fragment (TRF) analysis. Treatment with HAART blocked the accelerated telomere erosion normally seen in PBMCs during HIV infection (p<0.0001). Patients on HAART showed an average loss of 65 bcompared to the ~52 bp/year loss seen in age matched populations and the >180 bp/yr seen upon HIV infection in the absence of HAART. HAART blocks the accelerated telomere erosion seen in the circulating component of the immune system during HIV infection. However, it does not restore telomere sequences previously lost, indicating that repopulation of PBMCs from progenitor cells or activation of telomerase does not restore replication potenadvantage favoring earlier HAART use.

24

INCREASED ACCELERATION OF TELOMERE LOSS BY CHEMOTHERIN PATIENT

APY S WITH LOCALLY ADVANCED HEAD AND NECK CANCER

Brad Unryn, Desiree Hao, Stefan Glück, Linda Cook and Karl Riabowol (Riabowol Lab, University of Calgary) Chronic viral infection and treatment with combinations of chemotherapeutic drugs have been reported to accelerate the erosion of telomeres. In this study we asked if concurrent chemo- and radiotherapy, using the single agent cisplatin, would also accelerate telomere loss in head and neck cancer patients, and whether loss was linked to patient smoking habits, age, sex or stage of disease at diagnosis. We also asked if there was any alternation (?) in patient blood telomere length prior to treatment. While there was no evidence for telomere length differences prior to treatment, we found that chemoradiotherapy increased the rate of telomere erosion >100 fold. Although smokers had significantly shorter blood cell telomeres prior to therapy, smoking status did not affect chemotherapy induced telomere attrition, nor did sex or stage of disease at diagnosis. However, we did make the novel observation that a significantly greater loss of telomeres occurred in response to treatment in older patients, with those less than 55 years old losing an average of 400 base pairs (bp) of DNA compared to the 880 bp lost by patients 55 years and older. This difference was significant (p=0.025) suggesting that cisplatin may have more severe effects on the replicative capacity of blood cells in elderly patients.

25

Telomere and Telomerase Structure and Function

Chair: Chantal Autexier

26

ROLE OF TELOMERASE REVERSE TRANSCRIPTASE IN THE DIVERGENT EGULATION OF MOUSE AND HUMAN TELOMERASES AND TELOMERE

FUNCTION Fakhoury J.

R

, Marie-Egyptienne D. T., & Autexier C. (Autexier Laboratory, McGill University) Background: The telomerase ribonucleoprotein (RNP) is composed of a reverse transcriptase (RT) catalytic subunit (hTERT in human, mTERT in mouse), associated proteins, and an integral RNA component (hTR for human telomerase RNA, mTR for mouse TR) that provides the template for synthesis of telomeric (TTAGGG) DNA repeats. Marked differences exist in en e repeat-addition activity, ribonucleoprotein complex size and telomere length between human and mice. We hypothesize that some of th ouse may re oteins, an eover, we predi T since previ n is impli sis. Methods and Results: To address whether differences in the interaction of the various TERT and TR components may contribute to difference n the observed in vitro activities of reconstituted telomerase enzymes, we immunoprecipitated tagged-TERT components and analyzed coimmunoprecipitated TRs. Our re heterologous TERT-TR interactions occur, and thus the observed differences in enzyme activities cannot be explained by a lack of protein-RNA interactions. To address whether cellular factors are essential for the regulation of telomerase activity we expressed the various telomerase complexes in mouse or human cells, immunoprecipitated tagged-TERT components, and analyzed telomerase activity. Activities of the telomerase complexes expressed in cells paralleled the activities of the telomerase enzymes reconstituted in vitro, suggesting that there are no major differences in cellular fact s regulating telomerase catalytic function in vitro. Future Experiments: Functional differences in activities will be assessed by exchanging N-terminus, and assaying fo ere maintenance in vitro and in TERT.

zym

e distinctive features of telomerase and telomere biology among human and msult from species-specific differences in TERT-TR interactions, associated prd TERTs that are required for activity of human and mouse telomerases. Mor

ct that these differences reside in the N-terminus of hTERT and mTERous work from our lab and from other groups has shown that this regiocated in telomerase activity, TR binding, and telomere length homeosta

s i

sults indicate that

or

domains between hTERT and mTERT, specifically in ther telomerase activity, processivity, localization, and telom various mouse and human cell lines lacking either TR or

27

REGULATION OF TELOMERASE BY Rnt1p. Stéphanie Larose, Nancy Laterreur, Sherif Abou Elela & Raymund J. Wellinger

des the action of dsRNA endonucleases. They re part of the RNase III family and are implicated in the maturation of precursor rRNAs

romyces the re-

RNA

are

cells bundance

r

he d by

ht

d

2. Lebars I

(Wellinger Lab, Université de Sherbrooke) RNA metabolism in eukaryotic cells incluaand the processing of small RNAs (snRNAs and snoRNAs). In the yeast Sacchacerevisiae, the only known dsRNA endonuclease is called Rnt1p. In addition to above-mentioned functions, Rnt1p is also implicated in degradative pathways for pmRNAs and mRNAs and our lab recently showed it to be involved in a nuclear mdegradation mechanism1. In order to get better insight into the various RNAs that are affected by Rnt1p, we performed a comparative microarray analysis between a wt and a rnt1∆-strain. This analysis revealed that mRNAs encoding various subunits of the yeast telomerase specifically up-regulated in a rnt1∆-strain. Northern blot analysis confirmed that indeed, EST1, EST2 and EST3 mRNAs as well as the TLC1 RNA appear more abundant inlacking Rnt1p compared to wild type. Western blots confirmed an increased aof Est2p and there is also increased telomerase activity in the absence of Rnt1p. Finally, Rnt1p clearly has an effect on telomere homeostasis as telomeres in rnt1∆-cells appeaslightly longer than in wt. We know that Rnt1p cleaves RNA stem-loop structures in which the loop includes a conserved NGNN tetraloop2. Computer analysis identified a potential cleavage site in tmRNA of EST1 and in vitro analyses showed that EST1 mRNA is cleaverecombinant Rnt1p. Specific mutations predicted to abolish the consensus cleavage sitefor Rnt1p in the EST1 mRNA indeed abolished cleavage and resulted in a sligstabilization of the EST1 mRNA during late S phase of the cell cycle. Therefore, our datasuggest that the abundance of the EST1 mRNA is regulated directly by Rnt1p-mediatecleavage and this may have an impact on the other telomerase-encoding mRNAs. We arenow investigating the transcriptional mechanisms by which Rnt1p regulates telomerasemRNAs and the TLC1 RNA. References: 1. Ge D, Lamontagne B, Elela SA. Curr Biol. (2005) Jan 26;15(2):140-5.

, et al. EMBO J. 2001 Dec 17;20(24):7250-8.

28

THE ROLE OF THE KEOPS COMPLEX IN TELOMERE FUNCTION Michael Downey, Rebecca Houlsworth, Laura Maringele, Adrienne Rollie, Marc

rehme, Sarah Galici, Sandrine Guillard, Melanie Partington, Mikhajlo K Zubko, Nevan t, Lea Harrington, David Lydall and Daniel

urocher.

of five novel suppressors, including the previously

ns. We suggest a model whereby KEOPS regulates chromatin

BJ. Krogan, Andrew Emili, Jack F. GreenblatD (Durocher Lab, Samuel Lunenfeld Research Institute and The University of Toronto) Telomere capping is the essential function of telomeres. To identify new genes involved in telomere capping, we carried out a genome-wide screen in Saccharomyces cerevisiae for null suppressors of cdc13-1, an allele of the telomere capping protein Cdc13. We report the identificationuncharacterized gene YML036W, which we name CGI121. Cgi121 is part of a conserved protein complex – the KEOPS complex – containing the protein kinase Bud32, the putative peptidase Kae1, and the uncharacterized protein Gon7. Deletion of CGI121 or BUD32 suppresses cdc13-1 via the dramatic reduction in ssDNA levels that accumulate in cdc13-1 mutants. Deletion of BUD32 or other KEOPS components leads to a very dramatic short telomere phenotype and a failure to add telomeres de novo to DNA double-strand breaks. Cells deficient in Bud32 have wildtype telomerase activity in vitro and tethering of telomerase to the end of the chromosome does not rescue telomere-length defects in these straiarchitecture and remodeling at the chromosome end in vivo.

29

C-MYC ALTER THE THREE-DIMENSIONAL ORDER OF TELOMERES AND HROMOSOMES IN THE INTERPHASE NUCLEUS C

Sherif F. Louis1, Bart Vermolen2, Yuval Garini2 and Sabine Mai1 (Mai lab, University of Manitoba)

he ncoprotein c-Myc was found to be deregulated in over seventy percent of all cancers.

during subsequent cellular divisions leaving the daughter cells with non-reciprocal translocations and terminal deletions. The remaining broken uncapped chromosomes are highly reactive and engage in further fusion events leading to bridge-breakage-fusion (BBF) cycles 4. BBF cycles lead to the accumulation of genetic abnormalities. 1: Mai S et al. Oncogene 12(2):277-88.1996 2. Mai S et al. Chromosome Res (5):365-71.1996 3. Felsher DW and Bishop JM. Proc Natl Acad Sci (USA) 96(7):3940-4. 1999 4. Louis S et al. Proc Natl Acad Sci (USA) 102(27):9613-8. 2005

Deregulation of oncoproteins leads to genomic instability and tumorigenesis. ToExperimental deregulation of c-Myc, in vivo and in vitro, leads to the formation of karyotypic instability including amplifications, translocations, deletions, insertions, aneuploidy and polyploidy 1-3. However the pathway(s) by which such genomic abnormalities are formed is not fully understood. Here we studied mechanisms of chromosomal rearrangements, specifically unbalanced translocations and terminal deletions. We focused on the detailed events and their sequence upon the deregulation of c-Myc in diploid mouse PreB lymphocytes. We recorded the positions and volumes of telomeres and chromosomes respectively after Myc deregulation. We report that c-Myc induces telomeric aggregates and fusions followed by end-to-end fusions of chromosomes that involve both ends of mouse chromosomes. During the telomeric fusion events, the structure and architecture of chromosomes in the interphase nucleus is remodeled. Specific pairs of chromosomes are brought into closer proximity. Chromosomes being physically closer undergo repeated translocations and recombinations. Moreover, dicentric chromosomes break

30

Te

chniques in Telomere Biology

Chair: Raymund Wellinger

31

REAL-TIME PCR ASSESSMENTS OF TELOMERE LENGTH, TELOMERASE CTIVITY AND GENE EXPRESSION RESPONSE TO ECTOPIC TELOMERASE

Steven D. Perrault, W. Allan King and an H. Betts

A

De (Betts Lab, Department of Biomedical Sciences, University of Guelph) Recent evidence suggests that telome e might have roles other than telomere length extension. However, the conventional telomerase repeat amplification protocol (TRAP) is limited in its ability to accurately quantify telomerase activity, particularly in samples in which telomerase is present at very low levels or presumed absent. Here, we describe the optimization of Real-Time telomerase activity and telomere length assays for use on the Roche LightCycler that can be combined allowing for an accurate quantification of both telomerase activity and telomere length om the same sample. Transgenic introduction of

le 22 he

0.043. Global gene expression response to ectopic telomerase was examined by microarray analysis of 19,000 genes using a human cDNA array. A high stringency significance analysis of microarrays (SAM) cutoff revealed 110 up-regulated and 174 down-regulated genes in response to high te erase levels. Real-Time RT-PCR analysis supported the altered expression of developmental and pro-proliferative genes such as IGF-2 and TGF-β family members, tp51bp1, PAPPA, MET and MIA suggesting that telomerase function(s) may go beyond establishing the proliferatve capacity of cells through telomere maintenance/elongation to promoting the cellular proliferation and survival characteristics observed for such cells. Funding provided by CIHR and NSERC.

ras

frhuman telomerase (hTERT) into bovine adrenal cortical (AC) cells increased measurabtelomerase activity levels by approximately 17-fold, from 0.026 ± 0.002 to 0.440 ± 0.0(relative to 293T cells). As well, we found that upon introduction of telomerase, trelative telomere length ratio (to 293T cells) was increased from 0.74 ± 0.05 to 1.23 ±

lom

32

HUMANIZED YEAST AS A TOOL TO STUDY TELOMERE BIOLOGY Amadou Bah, Éric Gilson and Raymund J.Wellinger (Collaboration of the Wellinger and Gilson labs, Université de Sherbrooke and École

ormale Supérieure de Lyon, France)

[4] Berthiau, A.S. et al.; (2006). Embo J, 25, 846-856.

N Synthesis of vertebrate-type repeats at yeast chromosomal ends can simply be allowed by reprogramming the yeast telomerase RNA template region to a vertebrate template sequence [1]. Replacement of the original sequence by human telomeric DNA is progressive over the generations [2,3]. However, deep sequence replacement, regular T2AG3 repeats taking over TG1-3 repeats, is not without any cellular consequences. While the newly added sequence is maintained, such mutation of the template leads to an increase of single strandedness at telomeres [3], a different logic in the telomere length sensing mechanism [4], late anaphase accumulation of cells and telomere-telomere fusions. These particular phenotypes of humanized yeast cells can provide an unusual tool for studying the function of yeast and human telomere-binding proteins. In this regard, we address the role of hTRF2 in humanized yeast with an altered terminal end structure. References: [1] Henning, K.A. et al.; (1998). Proc Natl Acad Sci U S A, 95, 5667-5671. [2] Brevet, V. et al.; (2003) Embo J, 22, 1697-1706. [3] Bah, A. et al.; (2004) Nucleic Acids Res, 32, 1917-1927.

33

AN ATTEMPT TO PURIFY A TELOMERASE HOLOENZYME FROM S. CEREVISIAE Nancy Lévesque, Alain T. Dandjinou, Raymund J. Wellinger

n Saccharomyces cerevisiae, the telomerase RNA is called TLC1. This RNA has a

eferences: , A.T. et al.; (2004) Curr. Biol., 14(13), 1148-58.

and Cech, T.; (2004) Proc Natl Acad Sci U S A, 6;101(27),10024-9.

(Wellinger Lab, Université de Sherbrooke) Itemplate region for the addition of repetitive DNA at the end of linear chromosomes and also acts as a scaffold for the assembly of proteins into a ribonucleoprotein complex. In an effort to discover all proteins associating with TLC1 RNA, we added an RNA tag (S1 aptamer) to the full length telomerase RNA in two locations. The actual sites for tag-addition were chosen based on predicted exposed sites using the secondary-structure prediction for this RNA1,2. The introduction of the S1 aptamer on TLC1 RNA does not affect the function of telomerase as it has little effect on telomere length maintenance. However, the S1 aptamers have a high affinity for streptavidin, thus allowing the purification of the complex with high selectivity using streptavidin-linked agarose beads. We follow the steps of purification by quantitative RT-PCR on the TLC1 RNA. I will present preliminary results of this purification process. R1. Dandjinou

2. Zappulla, D.C.

34

Yeast Telomerase

Chair: Lea Harrington

35

SEARCHING FOR GENES AND PATHWAYS INVOLVED IN A RAD52-, ELOMERASE-INDEPENDENT MECHANISM OF SURVIVAL IN YEAST.

Catherine Le Bel

T

, Emanuel Rosonina, Laura Maringele, David Lydall, and Lea Harrington. (Harrington Lab, University of Toronto and Lydall Lab, University of Newcastle) Telomeres play crucial roles in protection of linear chromosomes and genome stability. Their maintenance usually depends on the action of telomerase, the enzyme composed of two essential subunits: Est2 (the telomerase reverse transcriptase) and TLC1 (the telomerase RNA), together whose role is to replenish the ends of chromosomes. In yeast cells, the absence of telomerase function leads to an EST phenotype. This phenotype is characterized by the loss of telomeric sequences with increasing number of cell divisions, and culminat 0 to 80 generations. Lundblad sho scapes as ‘survivors’, a process p in mammalian cells

undblad, V. and Blackburn, E. H., Cell, (1993)). Until now, two major pathways for generation of survivors in absence of telomerase have been reported and studied, and th pathways appear to depend on the recombination protein Rad52 (Le, S. et al., netics, (1999); Teng, S. and Zakian, V.A., Mol. Cell. Biol., (1999); Teng, S. et al., l. Cell, (2000); Chen, Q. et al., Mol. Cell. Biol., (2001)). . rtain conditions were recently discovered under which yeast cells proliferate in the sence of telomerase and recombination. For example, Maringele and Lydall observed rvival of the yeast strain W303 in the absence of RAD52, TLC1, MRE11, and EXO1 aringele, L. and Lydall, D. Genetics, (2004); Maringele, L. and Lydall, D. Genes Dev.

4)). Telomeres in these ‘PAL survivors’ are comprised of unusual palindromes ed from non-telomeric chromosomal termini (Maringele, L. and Lydall, D., Genes

., (2004)). Surprisingly, we find that survivors may be isolated, albeit at a low uency, in T2 and RAD52 without

the apparent ns. We are currently characterizing ing the telomere length dynamics, in the est2∆rad52∆ survivors. These data suggest that eukaryotic cells may possess a surrogate telomere length maintenance mechanism that does not rely on RAD52 or the absence of MRE11 and EXO1.

es with the entry into senescence after approximately 6wed previously that a small subset of this population e

ossibly related to the ALT mechanism observed (LtheboGeMoCeabsu(M(200formDevfreq certain yeast strain backgrounds in the absence of ES

requirement for other pre-existing gene deletio this mechanism of escape from senescence, includ

36

TELOMERASE- AND CAP-INDEPENDENT SURVIVORS IN YEAST. Michel Larrivée, Victor Karpov and Raymund J. Wellinger

ab, Université de Sherbrooke)

lity. In irtually all organisms, the telomerase enzyme provides this function, but telomerase-

as Cdc13p, but can be

(Wellinger L Maintaining telomeric DNA at chromosome ends is essential for genome stabivindependent mechanisms also exist. These latter mechanisms rely on recombination pathways to replenish telomeric DNA and extrachromosomal DNA may be implicated. We will report that in S. cerevisiae cells, extrachromsomal circular DNA occurs for both subtypes of telomerase-independent telomere maintenance mechanisms. This DNA is comprised of circular molecules of full length subtelomeric repeat elements for the type I mechanism, and very heterogeneously sized and at least partially single-stranded circles of telomeric repeat DNA in type II cells. Surprisingly, both type I and type II cells canadapt to a loss of the normally essential telomere capping protein Cdc13p, inducing an alternate and reversible state of chromosome ends. Such cells lacking Cdc13p can still maintain chromosomes in a linear form, but some cultures also display clear evidence for chromosome circularization. Adaptation to this new way of maintaining chromosomes also involves an abrogation of the ability to induce DNA-damage checkpoints, leading to sensitivity of the cells to DNA-damaging agents. Chromosome capping therefore is not strictly dependent on the canonical capping proteins, suchachieved in an unconventional fashion.

37

FOLLOWING THE INTRACELLULAR PROCESSING AND LOCALIZATION OF THE YEAST TELOMERASE RNA. C. Olivier1, F. Gallardo1, R.J. Wellinger2 and P. Chartrand1

in a nucleolar accumulation of this RNA. On the other hand, deletion of Ku70 or EST2 leads to cytoplasmic and perinuclear localization of TLC1 RNA, respectively. Deletion of the Ku-binding stem in the TLC1 RNA also results in a cytoplasmic accumulation of this RNA, underlining the importance of KU in the trafficking of the yeast telomerase. Altogether, these results suggest that the maturation and assembly of the telomerase occurs in several intracellular compartments. By combining these techniques upon endogenous conditions, we have been able to elucidate parts of the maturation process and wish to address a complete view of the maturation and the trafficking of the TLC1 RNA.

1Laboratory P. Chartrand, Département de Biochimie, Université de Montréal, Québec, H3C 3J7. 2Département de Microbiologie, Université de Sherbrooke, Sherbrooke, Québec, J1H 5N4.

Telomeres are constituted of DNA repeats and protein capping complexes involved in protecting the genome from degradation and non-homologous end joining (NHEJ). Maintenance of telomeres length is assured by the telomerase holoenzyme which, in yeast, is composed of at least four proteins named EST and an RNA component named TLC1. While the maturation and assembly of the telomerase holoenzyme is currently well under study, little is known about its intracellular trafficking. Therefore, we have used an in situ hybridization technique that allows us to detect the endogenous form of TLC1 RNA in order to determine the intracellular location of its maturation, processing and recruitment to telomeres. Our results show that the TLC1 RNA is detected in 7 to 10 foci inside the cell nucleus, a number concomitant with a normal clustering of yeast telomeres. We also found that the deletion of the cap methyl-transferase TGS1, which modifies the cap of TLC1, results

38

D

NA damage and repair and

telomere length maintenance

Chairs: Tara Beattie and Susan Lees-Miller

39

BRCT DOMAINS – CONSERVED PHOSPHO-PRPTIDE RECOGNITION ODULES IN THE DNA DAMAGE RESPONSE

J.N. Mark Glover

M

(Department of Biochemistry, University of Alberta, Edmonton, AB) The C-terminus of BRCA1 contains a pair of tandem sequence repeats termed the BRCT (BRCA1 C-terminal) region. Structural studies from our group and others have shown that the two repeats pack in a head-to-tail manner to form a single protein domain that functions to specifically recognize and bind phospho-peptides containing the sequence motif pSer-x-x-Phe. This function is responsible for the interaction of BRCA1 with its biological partner proteins, such as the BACH1 helicase, to ultimately regulate the cell cycle in response to DNA damage. Mutations in the BRCA1 BRCT region that have been

ing the

olved hown of the

histone variant, γ-H2AX, thereby recruiting MDC1 and other repair factors to the sites of DNA double strand breaks. In addition, the BRCA1 partner protein, BARD1 also contains a pair of BRCT repeats. Our str ture of this domain reveals a conserved phospho-serine recognition pocket, suggesting that this domain may also play a role in DNA damage response signaling and, potentially, tumour suppression. The importance of these interactions in the DNA damage ponse suggests that inhibitors of these interactions could provide routes to sensitize tumours to DNA-targeting therapies.

linked to breast and ovarian cancers specifically block this interaction, indicatimportance of this interaction for the tumour suppressor function of BRCA1. BRCT repeats are not only found in BRCA1, but in a number of other proteins invin the DNA damage response. For example, we and other groups have recently sthat the BRCT repeats of MDC1 specifically recognize the phospho-peptide tail

uc

res

40

TELOMERIC CHROMATIN IN DIFFERENTIATION AND DNA DAMAG

E.

ster O’Dor E and Peter M. Lansdorp

erry Fox Laboratory, BC Cancer Agency, Vancouver, BC, Canada)

Telomeres are genomic guardians, ensuring chromosomes remain stable and

n structure telomere function. First, we are employing ChIP (Ch

(T

separate entities throughout the life of an organism. Models of telomeres detail a complex molecular assembly of telomere binding proteins and associated factors into a three-dimensional T-loop/D-loop structure that shelters telomeres from attack by DNA damage checkpoints and thereby maintains their protective functions. What most models ignore is the role that nucleosomes and the associated histone code of post-translational modifications may have in telomere structure and function. In vertebrates, very little is known about telomere chromatin structure and how various histone modifications contribute to telomere maintenance.

We are exploring two approaches to study the contribution of chromatito romatin ImmunoPrecipitation) to

n from pluripotent state to differentiated state. Since ES cells lack some damage and surveillance checkpoints, we predict major differences in telomeric chromatin before and after differentiation.

Our second approach is to study the silent chromatin associated with telomeric repeats on maintenance of telomere length and expression of nearby telomeric genes. We are constructing an ES cell line in which an unstable yellow fluorescent protein is adjacent to telomeric repeats of an initially fixed length. The unstable YFP will serve as a reporter gene whose expression and repression can be correlated with histone modification, telomere length, and the changes in chromatin after differentiation. We will discuss our progress in studying these links between chromatin structure and telomere function.

assess the enrichment of specific histone modifications at telomeres. Since ChIP is a candidate-based approach, we are focusing on a specific hypothesis of telomere function. Though telomeres try to avoid being processed as DNA damage, paradoxically, several DNA damage checkpoint and repair proteins are recruited to telomeres and are involved in normal telomere length maintenance. We predict that telomeres normally exhibit hallmarks of damaged DNA during the cell cycle, and that these modifications are required for normal telomere maintenance. We are testing this hypothesis by ChIPping with antibodies that recognize histone modifications specific to damaged DNA known in vertebrates and yeast, and correlating to cell cycle stages. In addition, by using mouse ES cells, we will be able to study changes in telomeric chromatin after differentiatio

41

AUTOPHOSPHORYLATION OF DNA-PKCS CONTROLS THE ENDONUCLEASE ACTIVITY OF ARTEMIS BY REGULATING ACCESS TO

1, Nick Morrice3, enelope Jeggo and Susan P. Lees-Miller

DNA ENDS Yaping Yu1*, Aaron Goodarzi2*, Ruiqiong Ye1, Pauline Douglas

2** 1** P

1 Southern Laberta Cancer Research Institute, University of Calgary 2 Genomic Instability Unit, University of Sussex, UK 3 Protein Phosphorylation Unit, University of Dundee, Scotland, UK * and **: these authors contributed equally to this work In human cells, DNA double strand breaks (DSBs) are repaired by one of two main pathways, namely, homologous recombination repair and nonhomologous enjoining (NHEJ). The main players in NHEJ include the Ku70/80 heterodimer, the catalytic subunit

mplicated in NHEJ, namely the exo/endonuclease, Artemis. Here, we hav

double-stranded to single stranded transition. These results have implications ot only for NHEJ but also for the role of DNA-PK at telomeres.

of the DNA-dependent protein kinase (DNA-PKcs), and the XRCC4-DNA ligase IV complex. Current models for NHEJ predict that Ku binds to DSBs and recruits DNA-PKcs to form the active protein kinase holoenzyme, DNA-PK. This is followed by recruitment of the XRCC4-DNA ligase IV complex. Surprisingly perhaps, NHEJ proteins including DNA-PKcs and Ku are also found at telomeres and DNA-PKcs null rodent cells display telomeric end-to end fusions. Moreover, the protein kinase activity of DNA-PK is required not only for NHEJ but also to prevent telomeric end-to-end fusions. Our lab has asked the question, if the protein kinase activity of DNA-PK is required for NHEJ and preventing telomere functions, what are the physiological substrates of DNA-PK. We have shown that in vitro, DNA-PK phosphorylates itself, Ku70, Ku80, and XRCC4; however, only phosphorylation of DNA-PKcs is required for NHEJ. Specifically, we have shown that a cluster of autophosphorylation sites between amino acids 2609 and 2647 in DNA-PKcs is required for “remodeling” the DNA-PK complex and presenting the DNA ends to DNA ligases. Recently, we have examined phosphorylation of another protein that has been i

e identified DNA-PK phosphorylation sites in vitro and show that Artemis is phosphorylated at the same sites in vivo in response to DNA damage. We show that the DNA-PK phosphorylation sites in Artemis are not required for the endonuclease activity of Artemis, however, autophosphorylation of DNA-PKcs is required for the endonucleolytic activity of Artemis. We propose that upon assembly at the DSB, DNA-PKcs undergoes an autophosphorylation dependent conformational change that presents the DNA ends to Artemis in a way that facilitates cleavage of DNA with 5’ overhanging ends at then

42

HUMAN TRF2, TRF1 AND TIN2 ARE INVOLVED IN AN EARLDAMAGE RESPONSE.

Y DNA

aul BradshawP 1, Dmitri Stavropoulos1, Magan Trottier1, David Gilley2 and Stephen

Hospital For Sick Children and University of Toronto; 2University of Indiana)

A lesions that can lead to mutation, complex signaling

cycle checkpoints, and apoptosis. he ATM protein kinase plays a critical early role in activating these DNA damage

Meyn1.

1( DNA double-strand breaks (DSBs) are dangerous DNchromosome aberrations, cell death or cancer. Human cells usenetworks to sense DSBs, then activate DNA repair, cellTresponses. Normal telomeres do not trigger ATM damage reponses, in part due to their association with TRF2. We find that TRF2, along with TRF1 and TIN2, rapidly associates with induced DSBs in non-telomeric DNA and that TRF2 inhibits DSB activation of ATM-dependent damage responses. Using a laser microbeam to induce DSBs in human fibroblast nuclei we find that, within 10 seconds post-irradiation, endogenous TRF2, TRF1 and TIN2 form foci that tightly colocalize with photo-induced DSBs and with DSB-associated ATM foci. YFP-TRF2 accumulates at DSBs as fast as GFP-ATM, but GFP-ATM remains longer at DSBs. Ionizing radiation induces phosphorylation of TRF2 at Thr188, and TRF2-Thr188P associates with DSBs rather than normal telomeres. While ATM phosphorylates TRF1 and TRF2, ATM is not required for TRF1 or TRF2 to associate with photo-induced DSBs. Neither are the kinetics of TRF2 accumulation at DSBs affected by Thr188 or Ser368 phosphorylation, suggesting that phosphorylation occurs after TRF2 is at a DSB. The TRF2 response is dependent on its basic domain but is not affected by the absence of telomerase or DNA damage response proteins, including DNA-PKcs, the MRE11/Rad50/NBS1 complex and the Ku70, WRN and BLM repair proteins. To test if TRF2 affects activation of ATM damage responses, we over-expressed TRF2 and found that over-expression impairs production of phosphorylated ATM, H2AX and p53 following γ-irradiation and interfers with the accumulation of GFP-ATM at DSBs. Our results suggest that TRF2, TRF1 and TIN2 may interact, either independently or as a complex, with DSB-containing chromatin, provide evidence that TRF2 may compete with or attenuate ATM DSB responses, and implicate these telomeric proteins in novel DNA damage response.

43

DNA-PK AND TELOMERE MAINTENANCE: WHAT’S NEW ON THIS E

ND?

icholas S.Y. TingN , Brant Pohorelic, Yaping Yu, Susan P. Lees-Miller and Tara L.

epartment of Biochemistry and Molecular Biology, Cancer Biology Research Group,

deficient for functional DNA-PK show creased rates of telomere loss, accompanied by chromosomal fusions and

re currently characterizing the functional cellular significance of the interaction between

Beattie. (DUniversity of Calgary) Maintenance of telomere integrity in human cells requires the dynamic interplay between telomerase, telomere associated proteins, and DNA repair proteins. These interactions are vital to suppress DNA damage responses and changes in chromosome dynamics that can result in aneuploidy or other transforming aberrations. One of the DNA repair proteins implicated in this process is DNA-PK (DNA dependent protein kinase). DNA-PK, which is composed of the catalytic subunit DNA-PKcs, and the dimeric DNA binding regulatory subunits Ku70/80, is required for repair of double strand breaks via the non-homologous end-joining pathway. Cells intranslocations. Moreover, treatment of cells with specific DNA-PK kinase inhibitors leads to similar phenotypes. The interaction between the Ku (yKu70/80) and the RNA component of telomerase (TLC1) in S. cerevisiae has been shown to be important for maintaining telomere length. Although there is no sequence similarity between TLC1 and the human RNA component (hTR) of telomerase (hTERT), we previously showed that human Ku70/80 interacts with hTR both in vitro and in a cellular context. Specifically, Ku70/80 preferentially interacts with a 47 nucleotide region at the 3’end of hTR, which resembles the stem loop region of yKu70/80 binding domain on TLC1. Together our data suggest Ku interacts directly with hTR, independent of hTERT. We aKu70/80 and hTR. Furthermore, we have identified a protein involved in telomere maintenance to be a target of DNA-PK phosphorylation. These data potentially reaffirms the importance of DNA-PK kinase activity in telomere maintenance, and may provide further insights into the function of DNA-PK at telomeres.

44

RAD50 ANTAGONIZES THE ACTION OF TRF1 ON HUMAN TELOMERE LENGTH CONTROL

Shujie Xiao and Xu-Dong Zhu Yili Wu, .

uman telomere length homeostasis is a critical determinant for tumorigenesis and aging.

(Zhu Lab, McMaster University) HHuman telomeres are associated with many DNA repair complexes including that of Rad50, Mre11 and Nbs1 proteins, but the precise role of this repair complex in telomere length maintenance is unknown. Here we show that Rad50 controls telomerase-dependent telomere elongation by modulating the association of telomeric DNA with TRF1, a negative regulator of telomere length maintenance. We found that wild type Rad50 and Rad50 mutants can be overexpressed at telomeres through their fusion to a telomere specific protein, hRap1. The properties of Rad50 are preserved in the context of the fusion proteins. We show that the fusion protein containing wild type Rad50 promotes telomerase-mediated telomere elongation whereas the fusion proteins containing loss of function mutations in Rad50 fail to induce telomere lengthening. Our results identify human Rad50 as a positive mediator for telomerase-dependent telomere synthesis as previously shown for yeast Rad50, implying a conserved function of Rad50 in telomere length maintenance between yeast and human. Furthermore, we show that physical association of TRF1 with telomeric DNA is significantly reduced in cells expressing the fusion protein containing wild type Rad50, indicating that Rad50 acts as an antagonist of TRF1 in governing the human telomere length control. We propose that Rad50 and TRF1 are opposing forces modulating the state of telomeres needed for the action of telomerase.

45

POSTERS

46

TOWARDS TELOMERE ELONGATION USING TRUNCATED HUMAN PROTECTION OF TELOMERES 1 (hPOT1) PROTEINS Geraldine Aubert1, Irma Vulto1, Mike Schertzer1 and Peter M. Lansdorp1, 2

1Terry Fox Laboratory, British Columbia Cancer Agency,Vancouver, BC, Canada. 2Department of Medicine, University o ritish Columbia, Vancouver, BC, Canada

Genome maintenance and replicative lifespan in cells requires conservation of telomeric DNA and telomere associated protein complexes at chromosome ends. Human protection of telomeres 1 or hPOT1 binds single stranded telomeric (TTAGGG) DNA repeats and is thought to act as both a capping mechanism and a sensor of telomeric DNA length, regulating telomere length maintenance and elongation.

hPOT1 plays a role in the m intenance of telomeres of all cells. A deletion mutant of the hPOT1 protein - lacking DNA binding regions - was shown to induce rapid telomere elongation when over-expressed. We are exploring the effect of TAT-hPOT1∆ proteins transduced into primary immune cells. Transduced cells were tested to determine effects on the average cellular telome length by quantitative flow-FISH. Preliminary results show modest telomere elongation in primary human lymphocytes and we are developing new techniques combining fluorescent cell tracking and Flow-FISH to differentiate between resting and dividing cells to improve the sensitivity in short term culture measurements. In addition, we are also testing similar hPOT1∆ constructs using a retroviral transduction xpression of hPOT1∆ proteins in Jurkat cells (human T ce

Poster 1

f B

a

re

system to obtain sustained ell line) and primary T cells.

47

INVESTIGATING THE CELL SURVIVAL ROLE OF TELOMERASE IN TIVE

A. and Autexier, C

HUMAN NEURONAL CELLS: IMPLICATIONS FOR NEURODEGENERADISEASES Jain, P., Demers, J., Zhang, Y., Giannopoulos, N.P. Hammond, J., LeBlanc,

.

Backgro

that occur during aging include t

ell lines. We found that the differentiated cells express neuron-specific markers, cluding glutamate receptor and tau protein, similarly to primary human neurons. ifferentiation represses telomerase activity and human TERT expression paralleling the lomerase status of primary human neurons. Conclusions and Future Experiments: euronal markers and telomerase status are similar in differentiated human neuronal NT2

nd SK-N-SH cells and primary human neurons. These results validate the use of the ifferentiated NT2 and SK-N-SH cells to investigate the cell survival role of telomerase. TERT will be expressed in differentiated NT2 and SK-N-SH cells, and primary human eurons, and the effects on cell survival in the presence or absence of cell stresses linked Alzheimer’s disease (amyloid β-peptide, Fe2+) will be investigated.

(Autexier Laboratory, McGill University)

und: Life expectancy in Canada has nearly doubled during the 20th century, from <50 years of age in 1900, to 80 years of age in 1990. The consequence of longer life expectancy is the increased risk of exposure to age-related disorders, including neurodegenerative diseases, such as Alzheimer’s, and the associated healthcare related costs. Molecular mechanisms mediating the cellular changes

elomere dysfunction, oxidative stress and cell death. Dysfunctional telomeres may contribute to the development of aging phenotypes, such as vascular disease, poor wound healing, and immunosenescence. Recently, an association between telomere length in blood cells and mortality in people aged 60 years or older was reported. Psychological stress is significantly associated with higher oxidative stress, lower telomerase activity and shorter telomere length. Our hypothesis is that survival-promoting proteins such as telomerase, could ultimately affect brain cellular survival. Our studies could lead to novel therapeutic approaches for restoring neuronal function in various neurodegenerative conditions like Alzheimer’s. Methods and Results: To assess the protective role of telomerase against cell stresses typically linked to Alzheimer’s disease, we are using primary human neurons and differentiated human neuronal cells.

e established improved differentiation protocols for the NT2 and SK-N-SH neuronal WcinDteNadhnto Poster 2

48

ASSESSMENT OF DAMAGE AT TELOMERIC DNA USING COMET-FISASSAYS

H

lie Brind’AmourJu 1, Judit Banáth2, Peggy Olive2 and Peter M. Lansdorp1, 3

x Laboratory, British Columbia Cancer Agency, Vancouver, V5Z 1L3, BC, anada.

h Columbia Cancer Agency, Vancouver, V5Z L3, BC, Canada.

1The Terry FoC2Department of Medical Biophysics, Britis13Department of Medicine, University of British Columbia, Vancouver, BC, Canada.

The Comet assay has been developed to study DNA damage at the single cell level. The assay is based on microscopic visualization of cells embedded in agarose, lysed and submitted to an electric charge to allow migration of DNA fragments and loops. Different types of DNA damage can be evaluated, such as single strand breaks, double strand breaks, DNA interstrand crosslinks and endonuclease-sensitive base damages. Unlike many other techniques used to estimate DNA damage, the Comet assay enables the observation of a heterogeneous response to genotoxic stress within a cell population. Moreover, it is possible to combine the Comet assay with antibody staining or FISH to study response heterogeneity within different areas of the genome.

We aim to utilize the Comet-FISH technique using telomere-specific PNA probes

to study specific DNA damage at or near the telomeres. We are testing different lysis and migration conditions that allow conservation of the interaction of the telomeres with the nuclear matrix while permitting migration of the damaged DNA.

Poster 3

49

CHARACTERISATION OF THE SECONDARY STRUCTURE OF A POTENTIAL PSEUDOKNOT IN THE TLC1 TELOMERASE RNA OF S.

EREVISIAE. C Annie D’Amours, Bruno Lamontagne et Raymund J. Wellinger

r Lab, Université de Sherbrooke)

synthesis of chromosome ends, the telomeres, depends on an enzyme verse

anscriptase subunit, Est2p, a number of other proteins, as well as an RNA moiety, TLC1 in yeas

NA of S. cerevisiae has not yet been firmly established. Therefore, our goal is to establish a second

Refs: . Dandjinou, A.T. et al. 2004. Curr Biol. 14: 1148-58. . Chen, JL. and Greider, CW. 2004. Proc Natl Acad Sci USA.

101:14683-4.

(Wellinge In eukaryotes, the called telomerase. This particular ribonucleoprotein is composed of a retr

t, which dictates telomere repeat addition. Results derived from many organisms have shown that the association of the telomerase RNA to the holo-enzyme relies much more on the secondary and tertiary structure of the RNA than sequence specificity. In particular, we recently obtained evidence suggesting the presence of a pseudoknot near the template region of the yeast telomerase RNA1. The region of this potential pseudoknot overlaps with the region of Est2p association and given the presence of a pseudoknot structure in or near the catalytic core of telomerase of many organisms, a pseudoknot could be a common structural theme important for some aspect of telomerase function2. However, the presence of the pseudoknot in the telomerase R

ary structure in this particular region of the RNA using biochemical approaches. We constructed a shortened version of the catalytic core RNA and use RNase mapping for this purpose. Here we will present the first mapping results derived from this approach and describe our future plans for this project.

12

Poster 4

50

EXAMINATION OF TELOMERE ORGANIZATION IN HUTCHINSOGILFORD PROGERIA SYNDROME CELLS

N-

ichelle DeckerM , Liz Chavez, Mike Schertzer and Peter Lansdorp

erry Fox Laboratory, British Columbia Cancer Agency, Vancouver, BC, Canada sity of British Columbia, Vancouver, BC,

anada

TDepartment of Medical Genetics, UniverC

Hutchinson-Gilford Progeria Syndrome (HGPS) is a premature aging syndrome caused by mutations in the nuclear matrix protein, Lamin A. Lamin A is found predominantly at the nuclear periphery but recently has been discovered to occur throughout the nucleus in a ‘nucleoplasmic veil’. A single nucleotide mutation (1824 C T), which causes a silent amino acid change in Lamin A has been found in the majority of cases. HGPS cells are characterized by misshapen nuclei, chromatin disorganization and accumulation of the mutant protein. In addition, it has been shown that cells from HGPS individuals have shorter telomeres compared to parents’ cells and age-matched controls.

As a part of the chromatin disorganization found in HGPS, it is possible that the telomere organization is also perturbed. In order to assess this, we are interested to determine the location of telomeres in the nucleus using immunofluorescence using antibodies specific for lamins, Telomeric Repeat-Binding Factor 1 (TRF1) and other known nuclear proteins. By comparing the nuclear localization in HGPS fibroblast cells and BJ fibroblasts (that express wild type Lamin A) it will be possible to determine if telomere organization is different in the progeria cells.

Poster 5

51

DNA DAMAGE RESPONSE IN MURINE EMBRYONIC STEM CELLS AFIBROBLASTS DEFICIENT IN mTert

ND

Natalie Erdmann and Lea Harrington. (Harrington Lab, Campbell Family Institute for Breast Cancer Research/Ontario Cancer

elomeres require protection to distinguish them from double strand chromosome breaks. Expose

orp, P.M., Collins, K. & Hahn, W.C. (2005) PNAS 102, 8222-8227).

.

Institute) T

d telomeres elicit the DNA damage response and, without repair, lead to chromosome fusions, which in turn drive breakage-fusion cycles and genomic instability. Proteins such as Trf2 and Mre11 are involved in both a telomere-specific and a generalized response to DNA damage, but it is unclear how these potentially separate processes are coordinated. It has been recently published that human fibroblasts lacking the telomerase catalytic subunit (hTERT) have an impaired DNA damage response that is independent of telomere length maintenance and is associated with an altered chromatin state (Masutomi, K., Possemato, R., Wong, J.M.Y., Currier, J.L., Tothova, Z., Manola, J.B., Ganesan, S., Lansd

In order to confirm and extend this finding, we are investigating the DNA damage response in murine cells lacking mTert. To date, we have examined the induction of gamma-H2AX and phosphorylated ATM, p53, and p53BP1 following exposure of mTert-/- murine embryonic fibroblasts and ES cells to either gamma irradiation or etoposide. Preliminary data suggest a very mild suppression of gamma-H2AX induction in one mTert-/- ES cell line at early passage (i.e. when telomeres are long), however further analysis in other mTert-/- ES cells and MEFs is ongoing Poster 6

52

DEVELOPMENT OF AN ASSAY FOR DETECTION OF OLIGONUCLEOTIDCLEAVAGE BY HUMAN TELOMERA

E SE

Linda Holland, Rena Oulton, and Lea Harrington

ting

point sequence associated with e (α)TI determinant of α-thalassemia (Morin, 1991, Harrington and Greider, 1991). is oligonucleotide, termed (-12/-25)X9 (5’-GGAGGTGGGTGGGGCGCGATACT-

’), was previously shown to be cleaved and extended by human telomerase (Oulton and arrington, 2004). Our assay is a multi-step protocol in which this substrate is first cubated with telomerase under standard elongation conditions and then annealed to a mplate oligonucleotide that can discriminate between cleaved and uncleaved substrate. multiple cycles of primer annealing and extension with Taq polymerase only a cleaved bstrate is detected. To date, our results indicate that the amount of (-12/-25)X9 bstrate that is cleaved by telomerase under standard elongation conditions is lower than e minimal detection limit of this primer extension method. The feasibility of this and

ther strategies will be presented.

(Harrington Lab, CFIBCR, Ontario Cancer Institute, University of Toronto) The use of single-stranded oligonucleotide substrates in standard telomerase elongation assays has demonstrated that human, ciliate, and yeast telomerases are all associated with a nucleolytic cleavage activity. The human-telomerase associated nuclease is able to cleave 3’ non-telomeric nucleotides from substrates with 5’ telomeric sequence and also substrates that form mismatches with the hTER template (Oulton and Harrington [2004] Mol. Biol. Cell 15:3244-3256; Huard and Autexier [2004] Nucleic Acids Res. 32: 2171-2180). This activity may assist in the proofreading and/or processivity of the enzyme. Additionally, it might enable telomerase to heal chromosomes by exposing telomeric (G-rich) sequence at a broken chromosome end. It is thought that the nucleolytic ability is inherent to the catalytic core components (hTERT and hTR) as it co-purifies with human telomerase from endogenous and recombinant sources. However, the precise structural and functional characterization of a human-telomerase associated nuclease has been limited by the lack of an oligonucleotide cleavage assay that is independent of elongation activity. We are currently testing various elongation-independent methods of detecnucleolytic cleavage by human telomerase. One strategy utilizes an oligonucleotide substrate derived from the human chromosome 16 breakthTh3HinteInsusutho Poster 7

53

AN SGA APPROACH TO DISCOVER cdc13-1 SUPPRESSORS. Victor Karpov and Raymund J. Wellinger

ellinger Lab, Université de Sherbrooke)

elomeres, the DNA-protein complexes at the end of eukaryotic chromosomes, are ssential for chromosomal stability. In yeast, the telomeric single-strand binding protein

(W TeCdc13p has multiple important roles related to telomere maintenance:

• telomeric “capping” – protection of telomeres by forming complexes with yKu70/80 and with Stn1p/Ten1p;

• positive regulation of telomeric replication trough interaction with Est1p, which is a part of telomerase;

• negative regulation of telomerase by the recruitment of telomere elongation suppressors Stn1p and Ten1p.

In an attempt to identify genes that are involved in the deleterious outcome of an absence of Cdc13p, we screened the yeast gene knock-out library for genes that could suppress the growth defect of cdc13-1 cells at 33°C. For this purpose, we performed an SGA array experiment. cdc13-1 was marked with the NatR selective marker in the query strain and we scored for the ability of double mutant haploids to grow at 33°C. Eventually, we hoped to find the elusive genes involved in telomere 5'-end processing (exonucleases). Based on the comparative analysis of growth properties of the strains (23°C vs 33°C), the initial screen identified up to 111 genes that displayed an apparent growth at 33°C. In order to verify these results, diploids were regenerated, sporulated, microdissected, and haploid double mutants cdc13-1 yfg∆ were isolated from 38 potential cdc13-1 suppressors. We again scored the growth properties of all double mutants at 23°C (control), as well as 30°C, 32°C and 37°C for up to 72 hours. Unfortunately, this verification failed to reproduce a suppression of the growth defect by any of the selected genes at any temperature. While disappointing, the results reemphasize that careful reexamination of large scale SGA approaches are indispensable before going on to more involved experimentation. Poster 8

54

MAKING & BREAKING THE HAIRPIN TELOMERES OF THE LYME ISEASE SPIROCHETE.

erri Kobryn

D K , George Chaconas

obryn Lab, Université de Sherbrooke)

usual plurality of chromoDNA telomeres are generated from a replicated intermeUnexpectedly, the telomere resolvase (ResT) has also been shown to catalyze telomere bregenom s. We have employed modified replicated

(K Spirochetes of the genus Borrelia include agents that cause Lyme disease and relapsing fever maladies. The Borrelia genome is characterized by an un

somal elements, most of which are linear and are terminated by covalently closed hairpin telomeres. These hairpindiate in a DNA breakage and reunion reaction referred to as telomere resolution.

akage and fusion reactions; reaction reversals that could contribute to the pattern of e plasticity noted for these organism

telomere, hairpin telomere and half-site substrates to investigate the properties of the telomere formation, fusion and breakage reactions performed by ResT. Our results point to the establishment of a specific ResT-ResT synapse between half-site or hairpin telomere-bound ResT molecules as being a critical step in licensing ResT for DNA cleavage. This pattern of reactivity could help restrain the telomere resolvase from destroying its own product and inducing extreme levels of genome instability. Poster 9

55

INHIBITION OF TELOMERASE AND DESTABILIZATION OF TELOMIN MOUSE CELLS

ERES

Marie-Egyptienne D.T. and Autexier C.

ells with ifferent telomere lengths, and (ii) the introduction of mu-mTR on the viability of lomerase-positive mouse cancerous cell lines, and TR knock-out mouse cells. For both rojects, we generated stable clones expressing the transgene (mTERT-DN or mu-mTR). he effects of the long-term expression of the transgenes on telomerase activity, telomere ngth, cell viability and chromosomal abnormalities will be monitored. The CB17, LYR

nd LYS cell lines, that we determined have a telomere length of 16, 10 and 5 Kb spectively, were chosen for the introduction of mTERT-DN. At present, the CB17 TERT-DN expressing clones do not show any abnormalities in cell viability compared the control clones. The generation of clones from transfection in the LYR and LYS cell nes is currently underway. We introduced mu-mTR in telomerase-positive (CB17) and lomerase-negative mTR knock-out (DKO301) cell lines. Interestingly, contrary to the troduction of mu-hTR in human telomerase-negative cells, which impedes the

stablishment of clonal populations, we were able to isolate DKO301 mu-mTR clones hich thus far do not demonstrate cell viability impairment.

(Autexier Laboratory, McGill University) All cancer cells are able to divide indefinitely, which necessitates proper telomere maintenance. Telomerase is one mechanism mediating telomere maintenance and is active in 85% of cancers. Several studies have validated anti-telomerase strategies. The expression of a catalytically-inactive dominant-negative mutant of the telomerase protein subunit (TERT-DN) in cancerous cells leads to telomerase inhibition and telomere shortening but results in cell death, although only in cells with short telomeres. Moreover, the introduction of a template-mutated telomerase RNA subunit (mu-TR) in human cancerous cells lead to decreased cellular viability and increased apoptosis. The validation of anti-telomerase therapies may ultimately require the use of rodent models, and thus it is essential to also understand the consequences of telomerase inhibition or telomere destabilization in mouse cells. No studies have comprehensively analyzed the effects of telomerase inhibition in mouse cells with different telomere lengths or reported on the consequence of the introduction of mutant sequences at mouse telomeres. We are currently studying the outcome of (i) mouse telomerase inhibition in mouse cdtepTlearemtoliteinew Poster 10

56

MAPPING TELOMERASE REVERSE TRANSCRIPTASE DOMAINS THAT CONTRIBUTE TO TUMORIGENESIS Graeme AM Nimmo, Ryan Ward, Maria A Cerone, and Chantal Autexier

(Autexier Laboratory, McGill University)

Traditionally, reactivation of telomerase during tumorigenesis was thought to be required mainly to impart an indefinite lifespan via maintenance of telomere length. Recently, however, several studies have suggested that telomerase may contribute to tumorigenesis via an additional mechanism that could be independent of its role in telomere lengthening. It is currently unknown which region(s) of telomerase contribute to tumorigenesis.

Mapping the telomerase domains which mediate tumorigenesis can be achieved using well characterized immortal cell lines that maintain their telomeres in the absence of telomerase: ALT cells (alternative lengthening of telomeres). The ALT cell line GM847, which lacks hTERT cannot form subcutaneous tumors in nude mice solely with the addition of constitutively active Ras; only cells expressing both activated Ras and hTERT are able to form tumors (Stewart et al., 2002). To identify the hTERT regions responsible for promoting tumor formation we have generated GM847 and VA13 ALT cell lines stably expressing a constitutively active Ras mutant, RasV12. VA13 cells will be used to determine if hTR is required for the role of telomerase in tumorigenesis. We have stably transfected various mutants of hTERT, from our hTERT mutant library, into the Ras-expressing ALT cell lines and the ability of each mutant to confer the tumorigenic phenotype will be determined. The tumorigenic capacity of each transfected cell line will be assessed in vitro by their ability to grow in hard agarose (concentrations > 0.6% agarose). Finally, the growth capacity of cell lines containing the region(s) of hTERT responsible for this tumorigenic phenotype will be confirmed by subcutaneously injecting these cells into mice and observing tumor formation. Pinpointing the regions of telomerase responsible for tumorigenesis may identify a novel mechanism involved in transformation and may lead to the development of a new family of anticancer therapeutics selective for tumour cells. Poster 11

57

THREE DIMENSIONAL ORGANIZATION OF TELOMERES IN HEREDITAAND NON-HEREDITARY BREAST CA

RY NCER

Panigrahi S1 1 2,3 2 4 2 ai S1

versity of Manitoba, MB; 2. Departments of iversity, QC; 3. Algorithme Pharma, Laval,

ns is frequently seen in cancer cells. This change finally leads to “sticky”

rderline increased intensity (P = 0.091). We also bserved a significant difference in the occurrences of telomere aggregates in BRCA1-/+ =16), BRCA2-/+ (n=14) and non-hereditary (n=11) patients (p<0.05).

, Maiti S , Brunet JS , Kotar K , Bégin LR , Foulkes WD , M

(1.Manitoba Institute of Cell Biology, UniOncology and Human Genetics, McGill UnQC; 4. Department of Pathology, Sacre Coeur Hospital, QC.) Telomeres consist of tandemly repeated (TTAGGG)n sequences at the chromosomal ends and are capped by a complex of proteins, named shelterin. This special architecture prevents the eukaryotic chromosomes from being viewed as double strand breaks. A loss of telomeric protection, secondary to accelerated telomere shortening and disruption in shelteri functionchromosomal ends and initiates chromosomal end-to-end fusions. The end result is formation of telomeric aggregates leading to genomic instability. Our studies using high performance three-dimensional (3D) fluorescent imaging technologies indicate that telomeres of normal cell nuclei are organized into non-overlapping territories, in a dynamic and cell cycle dependent manner. In contrast, studies on the 3D organization of telomeres in cancer cells demonstrated the presence of telomeric aggregates. BRCA1 is an important DNA repair protein and possibly plays a role in telomere protection. We hypothesize that both germ-line BRCA1 and BRCA2 mutations increase genomic instability due to impaired DNA repair and reduced protection at the telomeric ends. Thus there is an increased probability of unprotected telomeric ends that can initiate end-to-end fusions and could be used as a new marker for detecting early changes in breast cancer cells. Our data with BRCA1 mutation (HCC1937) and with BRCA2 mutation (Capan-1) bearing cell lines shows the presence of significantly larger telomere aggregates in comparison to the telomeres of MCF-7(wt) cells (p < 0.0001). We also studied biopsy samples from 46 patients with early and advanced hereditary (n= 30) and non-hereditary breast cancers (n= 16) along with surrounding normal areas. Results from this study indicate that a) BRCA1/2-related breast cancers have a skewed distribution of telomeric intensities ompared with controls and b) have boc

o(n

Poster 12

58

ANALYSIS OF THE TELOMERIC 3’ SINGLE-STRANDED OVERHANG IN THE YEAST S. CEREVISIAE. Christian Prud'homme & Raymund J. Wellinger (Wellinger Lab, Université de Sherbrooke) Chromosome ends are composed of specific protein-DNA components that allow their protection and continued cell divisions. The very terminal DNA structure includes an evolutionary conserved G-rich single-stranded 3’-overhang, called the G-tails. These G-tails allow proper formation of the telomere and their extension by telomerase. In the yeast Saccharomyces cerevisiae, the degenerate nature of the telomeric repeat sequences as well as the very short length of the G-tails complicate the study of the telomeric ends. Recently, we have shown that in yeast, constitutive G-tails are approximately 12-14 nucleotides long and that the MRX-complex can affect this configuration1. However, the molecular mechanisms of G-tail formation and maintenance are still to be characterized. We have also developed a technique based on modified primer extensions in order to measure, at the nucleotide level, the length of the 3’ overhangs in various conditions and mutants1. This method can yield information on the dynamics of the telomeric structure and, using a variety of mutants implicated in telomere length regulation or G-tail maintenance, we can investigate the relevance of the overhangs in telomere biology. My goal is to first optimize this challenging biochemical tool and then apply it to various outstanding questions, such as the precise length of the G-tails in late S-phase, an analysis of leading vs lagging strand synthesis and the effects of various mutants (yku, tel1 and others). Reference: 1 Larrivée et al; 2004, Genes Dev. Jun 15;18(12):1391-6 Poster 13

59

CHARACTERIZATION OF THE MAMMALIAN KEOPS COMPLEX Rachel K. Szilard, Roger Agyei, Sarah J. Galicia, Nadine K. Kolas, Sara Sharifpoor,

Institute, Toronto)

, hGON7 /Chr10 ORF49, and ESO3/ITBA2/LAGE3, respectively) also form a omplex that functions at telomeres. Co-immunoprecipitation of endogenous proteins

cells have shown that PRPK can be found in association with CGI-121 and complex in human cells. We

redict that if the function of the KEOPS complex is conserved in human cells, inhibition f KEOPS components should lead to phenotypes that are opposite to those seen by hibition of shelterin components i.e. telomere shortening, an increase in telomere

apping, and suppression of a telomeric DNA damage response. We are currently vestigating the interactions between the entire complement of human KEOPS proteins,

s well as assaying for the effects of modulating expression of KEOPS proteins on lomere biology.

Anne-Claude Gingras and Daniel Durocher. (Durocher Lab, Samuel Lunenfeld Research Access to the telomere end must be balanced to allow for the replication of DNA ends and/or the elongation of telomeres by telomerase, while preventing the ends of linear chromosomes from being inappropriately recognized as DNA double strand breaks. The mammalian shelterin complex (de Lange, 2005, Genes Dev. 19: 2100-2110) binds to telomeric TTAGGG repeats, preventing the chromosome ends from eliciting a cellular DNA damage response and also regulating the addition of repeats by telomerase. In a screen for suppressors of a telomere capping defect, our lab recently identified a protein complex in Saccharomyces cerevisiae – the KEOPS complex – that is a regulator of telomere function (Downey et al., 2006, Cell 124: 1155-1168). In yeast, deletion of KEOPS components results in short telomeres and a defect in the addition of telomeres de novo to DNA double-strand breaks (telomere healing), suggesting that in addition to its role in promoting telomere uncapping, the KEOPS complex is also important for elongation of telomeres. The yeast KEOPS complex includes a protein kinase (Bud32), a putative endopeptidase (Kae1), and other proteins of as yet uncharacterized function (Cgi121, Gon7, and Pcc1). Since the mechanisms of telomere regulation are well conserved among eukaryotes, we hypothesize that the homologous human proteins (p53-related protein kinase (PRPK), hKAE1/OSGEP (O-sialoglycoprotein endopeptidase), CGI-121cfrom 293T with hKAE1, suggesting that these proteins do form a poincinate Poster 14

60

CELL CYCLE STUDIES ON THREE-DIMENSIONAL TELOMERE RGANIZATION. O

Landon Wark and Sabine Mai. (Manitoba Institute of Cell Biology, Winnipeg, CANADA) Telomeres are 3’ TTAGGG 5’ sequences protecting the ends of chromosomes from degradation. It was the purpose of our studies to analyze the three-dimensional (3-D) organization of telomeres during all phases of the cell cycle. It has been shown previously that varying telomere organizations are observed during different phases of the cell cycle with telomeric disks forming in the G2 phase of the cell cycle [1],[2]. The 3-D organization of telomeres demonstrates a highly ordered, nonrandom assembly of individual telomeres in the interphase nucleus. Mouse Pre-B lymphocytes were synchronized in late G2 with 0.5 ug/mL of nocodazole and in G1/S boundary with a combination of depleted media and normal media with mimosine and then fixed with 3:1 methanol/acetic acid. At the same time, samples were taken for sorting via Fluorescent Activated Cell Sorting (FACS), and stained with propidium iodide to confirm synchronization. Synchronized, unfixed cells were re-introduced into culture and harvested immediately after re-introduction and again after 8 hours. The harvested cells were stained using Quantitative Fluorescent In Situ Hybridization (Q-FISH) with a Cy3 PNA telomere probe and DAPI counterstain. The cells were imaged using Axiovision 4.3 and deconvolved using a constrained iterative algorithm. The data obtained in our study show that organization of the telomeres becomes disk-like during arrest in G1/S and G2/M checkpoints, but that that disk begins to lose its structure with the continuation of the cell cycle. 1. Chuang TCY., M.S., Garini Y., Chuang AYC., Young IT., Vermolen B., vanDoel R,Mougey V., Perrin M., Braun M., Kerr PD, Fest T., Boukamp P. and Mai S., The three-dimensional organization of

telomeres in the nucleus of mammalian cells. BMC Biology, 2004. 2(12): p. 1741-7007-2-12.

2. Vermolen B., G.Y., Mai S., Mougey V., Fest T., Chuang TCY., Chuang AYC., Wark L., Young I. T., Characterizing the three-dimensional organization of telomeres. Cytometry, 2005. 67A(2): p. 144-150.

Poster 15

61

XPF-ERCC1 IS REQUIRED FOR TRF2 MEDIATED TELOMERE SHORTENING Yili Wu, Natalia J. Zacal, Andrew Rainbow and Xu-Dong Zhu

(Zhu Lab, McMaster University) TRF2, a telomere binding protein, plays a crucial role in both telomere protection and telomere length maintenance. Overexpression of TRF2 results in telomere shortening, delaying the entry of human primary fibroblasts into senescence. TRF2 is found to be associated with XPF-ERCC1, a structure-specific endonuclease important for nucleotide excision repair, crosslink repair and DNA recombination. We found that TRF2-mediated telomere shortening is abrogated in cells deficient in XPF-ERCC1, suggesting that XPF-ERCC1 is required for TRF2-dependent telomere shortening. To further understand the role of XPF in TRF2-dependent telomere shortening, we generated constructs either containing wild type XPF or mutant XPF proteins carrying mutations in its conserved nuclease domain. We show that wild type XPF can complement XPF-deficient cells for UV repair whereas the mutant XPF fail to rescue the inability of XPF-deficient cells in UV repair, indicating that the XPF mutant proteins are defective in the nuclease activity required for nucleotide excision repair. However, when ovexpressed in XPF-deficient cells, both wild type XPF and mutant XPF proteins were able to partially rescue TRF2-dependent telomere shortening. Taken together, our results indicate that amino acids of XPF important for its function in UV-repair appear to be dispensable for its role in TRF2-mediated telomere shortening.

Poster 16

62

CHARACTERIZING INTERACTIONS BETWEEN HUMAN TELOAND TELOMER

MERASE IC PRIMERS IN VITRO

Haley D.M. Wyatt, Deirdre A. Lobb, and Tara L. Beattie. (Department of Biochemistry and

niversity of Calgary) Molecular Biology, Cancer Biology Research Group,

some healing. We believe that human telomerase has ifferent requirements for telomere binding versus telomere elongation, and so, have eveloped an in vitro binding assay to directly ascertain the protein and DNA equirements for a physical interaction between telomerase and telomeric oligonucleotide ubstrates. We have coupled this with the conventional telomerase extension assay to easure telomerase activity and repeat addition processivity. We are currently using this

ystem to characterize the requirements for physical and functional interactions between lomeric DNA primers and telomerase that has been reconstituted in vitro.

U Telomerase is a ribonucleoprotein complex that synthesizes telomeric repeats onto the ends of linear chromosomes. This enzyme has a critical role in maintaining telomere integrity, which is essential for an organism’s genetic stability and viability. During RNA-dependent telomere elongation, human telomerase hybridizes to the telomere through the complementary RNA template region and hTERT catalyzes the de novo addition of telomeric repeats via reverse transcription. Subsequently, the enzyme translocates to the newly synthesized chromosome end where the RNA template is realigned for another round of telomere synthesis. A unique feature of telomerase is that it accomplishes this task processively; this implies that translocation and realignment proceeds without enzyme dissociation from the telomere. One explanation is that telomeric DNA makes stable interactions with a telomerase “anchor site” domain, which is independent of the catalytic domain, which enables telomerase to translocate along the telomere without completely dissociating the enzyme/DNA complex after each round of telomere synthesis. Similarly, by imparting stability to an otherwise energetically unfavorable enzyme/DNA hybrid, an anchor site-DNA interaction helps to explain why telomerase can engage in RNA-independent telomere recognition and elongate non-telome NA during chromoric Dddrsmste Poster 17

63

ANALYSIS OF TELOMERE HEALING OF DNA DOUBLE STRAND BREAKS

ei ZhangW and Daniel Durocher

Durocher lab, Samuel Lunenfeld Research Institute, University of Toronto)

ks (DSBs) can be repaired mainly by homologous recombination R) or by non-homologous end joining (NHEJ). However, DSBs can also be “healed”

( DNA double strand brea(Hby their conversion into telomeres, a phenomenon referred to as telomere healing or de novo telomere addition. Stabilization of DSBs by telomere healing has been observed in many organisms, from budding yeast to human cells. Genetic studies, mainly in yeast, have shown that telomere healing requires a group of proteins that function either in DSB repair or telomere biology, including Mre11 nuclease, Cdc13, Ku70/80 heterodimer and telomerase. On the other side of the spectrum, the helicase Pif1 appears to be a negative regulator of this process. Recent studies from our laboratory indicate that telomere healing is more complex, as it has been showed that Bud32, a conserved protein kinase, is essential for telomere healing in Saccharomyces cerevisiae. To identify novel components required for telomere healing in eukaryotes I initiated a genetic screen in S. cerevisiae by employing a telomere healing reporter strain which possesses two counter-selectable markers (CAN1 and URA3) at a telomere-proximal location on chromosome V, along with a deletion of PIF1 gene. The PIF1 gene deletion causes a high rate of gross chromosomal rearrangements (GCRs) almost exclusively by telomere healing. However, if an essential telomere healing gene (e.g. YKU70) is also deleted, the GCR rate decreases to wild type levels. I am currently working to identify yeast strains whose GCR rate is reduced to wild-type levels and future work will be aimed at characterizing candidate genes for their roles in telomere healing of DSBs. Poster 18

64

Participants List Dr. Chris Counter

Duke University Medical Center C225 LSRC Box 3813 Durham, NC 27710 Tel: 919-684-9890 E-mail: count004@mc.duke.edu

Autexier Lab

McGill University Bloomfield Centre for Research in Aging Sir Mortimer B. Davis Jewish General Hospital 3755 Cote Ste. Catherine Road Montreal, Quebec H3T 1E2 Tel: 514-340-8222 ext. 4651 (office) Tel: 514-340-8222 ext. 5260 (lab) Fax: 514-340-8295 Email: chantal.autexier@mcgill.ca

Chantal Autexier Johans Fakhoury D.Tamara Marie-Egyptienne Graeme Nimmo

eattie Lab Department of Biochemistry and Molecular Biology University of Calgary 3330 Hospital Drive NW, HMRB 372B Calgary, Alberta T2N 4N1 Tel: 403-220-8328 (office) Tel: 403-210-9380 (lab) Fax: (403) 283-8727 E-mail: tbeattie@ucalgary.ca

B

Tara Beattie

Deirdre Lobb Brant Pohorelic Nicholas Ting Haley Wyatt

65

Betts Lab Department of Biomedical Sciences

ry College

ext. 54480 0

lph.ca

Ontario VeterinaUniversity of Guelph Guelph, Ontario N1G 2W1 Tel: 519-824-4120

-767-145Fax: 519Email: bettsd@uoguehttp://www.ovc.uoguelph.ca/biomed/Faculty/betts.shtm

Dean Betts

Chan L

Institute , Alberta

erboard.ab.ca

ab

Department of Oncology University of Alberta 4316, Cross CancerEdmontonT6G 1Z2 Tel: 780-432-8773(office) Email: gordonch@canc

Chaconas Lab

Department of ecular Biology Universi3330 Ho W, HMRB 272B

Alberta

ucalgary.ca

Jakub Famulsky

Biochemistry and Mol

ty of Calgary spital Drive N

Calgary, T2N 4N1 Tel: (403) 210-9692 Fax: (403) 270-2772 E-mail: chaconas@

George Chaconas Chartr

etit MontréaH3C 3J7Tel: 514Fax: 514Email: p hartrand@umontreal.ca

and Lab Département de Biochimie Université de Montréal 2900 Edouard-Montp

l, Quebec

-343-5684 -343-2210

.c

66

Pascal Chartrand Franck Gallardo

Catherine Olivier Done

ss Margaret Hospital twork

Medicine and Pathobiology

0-717, 10th Floor OCI/PMH, 610 Univ e, Toronto, ON, M5G 2M9

16-946-2337

ca

Lab Ontario Cancer Institute/PrinceUniversity Health NeDepartments of Laboratoryand Medical Biophysics, University of Toronto 1

ersity Avenu

Tel: 4Fax: 416-946-6579

toronto.E-mail: sdone@uhnres.uhttp://www.uhnres.utoronto.ca/donelab

riborz Ras Fa hid-Kolvear

Samuel Lunenfeld Research Institute oom 1073

Toronto, Ontario

Durocher Lab

600 University Avenue r

M5G 1X5 Tel: 416-586-4800 ext.4800 x2544 Fax: 416-586-8869 E-mail: durocher@mshri.on.caLab website: http://www.mshri.on.ca/durocher/

her Daniel Duroc

y

Wei Zhang

Sciences Building

ualberta.ca

Michael DowneRachel Szilard

olver Lab G

474 MedicalUniversity of Alberta Edmonton, Alberta T6G-2H7 Tel: 780-492 2136 Fax: 780-492 0886 Email: mark.glover@http://gloverlab.biochem.ualberta.ca/

67

Mark Glover Harring

ity of Toronto /University Health Network

bell Family Institute for Breast Cancer Research

lab)

i.utoronto.ca

ton LDept. Medical Biophysics, Univers

ab

Ontario Cancer InstituteCamp620 University Avenue, rm. 932 Toronto, Ontario M5G 2C1 Tel: 416-946-2834 (office) Tel: 416-946-4501 ext. 5569 (Fax: 416-204-2277 Email: lea.harrington@oc , or lea.harrington@uhnres.utoronto.ca

Lea Harrington Catherine Le Bel M Taboski

Hendz

ta s Cancer Institute

Natalie Erdmann Linda Holland

Marie Meznikova ichael

el Lab Department of Oncology University of Alber3332, CrosEdmonton, Alberta T6G 1Z2 Tel: 780-432-8439 (office) Email: michael.hendzel@ualberta.ca

Michael Hendzel Kobryn Lab

Département de microbiologie et d'infectiologie Faculté de médecine et des sciences de la santé

ité de Sherbrooke

64-5323

USherbrooke.ca

Univers3001, 12e Avenue Nord Sherbrooke, Québec J1H 5N4 Tel: 819-5Fax: 819-564-5392 Email: Kerri.Kobryn@ Kerri Kobryn

68

Lansdo p Lab

boratory

77 0712

r

Terry Fox LaB.C. Cancer Research Center 675 West 10th Avenue Vancouver, B.C. V5Z 1L3 Tel: 604 675 8135Fax: 604 8Email: plansdor@bccrc.ca

Peter M. Lansdorp

Julie Brind'Amour

Mike Schertzer

k Lees-

-220-7628

Geraldine Aubert

Liz Chavez Michelle Decker Maloy Ghosh Mark Hills Kathleen Lisaingo Ester O'Dor

Evert-Jan Uringa Irma Vulto Lindsey Laycoc

Miller Lab University of Calgary Biochemistry & Molecular Biology 3330 Hospital Drive NW 2894 Health Sciences Calgary, Alberta T2N 4N1

Tel: 403Fax: 403-283-4841 Email: leesmill@ucalgary.ca

er

Susan Lees-Mill

andon Cai iebert

Chris Williamson

Angela Zolner

Laura Baxter BrStephanie HChris KnightTing Li

Yaping Yu

69

Harry Zhou

Lefebics

Columbia h Columbia

822-5310 an e.ubc.ca

vre Lab Department of Medical GenetThe University of BritishVancouver, BritisTel: 604-Fax: 604-822-5348Email: louisl@interch g

Louis Lefebvre Mai Lab Manitob ology 675 McDermot Ave. Room ON6046 Winnipe R3E 0V9 Tel: 204 (office) Tel: 204 Fax: 204 Email: sm a.ca

a Institute of Cell Bi

g, MB

-787-2135-787-4127 (lab) -787-2190 ai@cc.umanitob

Sabine Mai

Soumya Panigrahi Sen

Martin

ily Institute for Breast Cancer Research e., Room 9-409

ntario

ext. 5290

Sherif Luois

AaronLandon Wark

Lab Scientist, Campbell Fam610 University AvToronto, OM5G 2C1 Tel: 416-946-4501, Fax: 416-946-2024 Email: lmartin@uhnres.utoronto.ca

Lisa Martin Mason Lab

Genome oratory Laval University Cancer Research Center Hôtel-D UQ) 9, McMahon street Québec

stability lab

ieu de Québec (CH

(Qc)

70

G1R 2J6Tel: (418) 525-4444 ext 15154 (office)

525-4444 ext 16816 (lab)

aval.ca

, Canada

Tel: (418) Fax: (418) 691-5439 E-mail: Jean-Yves.Masson@crhdq.ul

Jean-Yves Masson rance

Genetics and Genomic Biology pital for Sick Children

Matthieu Laf

Meyn Lab

HosRm 15-307 TMDT East 101 College Street Toronto, Ontario M5G 1L7 Tel: 416-813-8485 Fax: 416-813-4931 Email: meyn@sickkids.ca

Stephen Meyn haw

Heather Morin Lab

Michael e Sciences Centre British Columbia Cancer Agency

0 - 570 West 7th Avenue

4 7-5800 (admin)

ca

Paul BradsRoot

Smith Genom

Suite 10Vancouver, British Columbia V5Z 4S6 Tel: 604-707-580Tel: 604-70Fax: 604-876-3561 Email: gmorin@bcgsc.

Gregg Morin

iabowol Lab istry & Molecular Biology

ty of Calgary

8695

R

BiochemFaculty of Medicine, Universi#370 HMRB, 3330 Hospital Dr. NW Calgary, Alberta T2N 4N1 Tel: 403-220-

71

Fax: 403-270-0834 Email: karl@ucalgary.cahttp://www.ucalgary.ca/riabowol_lab/

Karl Riabowol Pinaki Bose

Xijing Han Mohamed Soliman Keiko Suzuki

Squire

ospital - ONTARIO CANCER INSTITUTE

rsity Avenue

ential voice mail) 01 ext. 5012 (lab)

Fax: 416 fidential) Email: je oronto.ca

Linda Cook Annie Fong

Brad Unryn

Lab Princess Margaret HRoom 9-721 610 UniveToronto, Ontario M5G 2M9 Tel: 416-946-4509 (confidTel: 416-946-45

-946-2840 (conremy.squire@ut

Web: http://www.utoronto.ca/cancyto/Tel: 416-946-2806 (office - Mark Brownell)

mbrownel@uhnres.utoronto.caEmail: Bisera Vukovic

Anthony Joshua Wellinger Lab

y and Infectious Diseases ité de Sherbrooke

Sherbrooke, Quebec

Tel: 819-564 5214 4 5392

brooke.ca

Dept. of MicrobiologFaculty of Medicine; UniversRm 8428 3001 12e Ave Nord

J1H 5N4

Fax: 819-56Email: Raymund.Wellinger@UsherWeb: http://callisto.si.usherb.ca/~microbio/wellinger/

Raymund J. Wellinger Amadou Bah

rs Annie D'AmouVictor Karpov

72

Nancy Laterreur

Christian Prud'Homme Zhu Lab

Departm ogy, LSB 438 McMaster University 1280 Ma t HamiltoL8S 4K1

xt. 27737 Fax: 905-522-6066

@mcmaster.ca

Nancy Lévesque Jean-François Noël

ent of Biol

in St. Wesn, Ontario

Tel: 905-525-9140 e

Email: zhuxu Xu-Dong Zhu

Yili Wu

73