Telomeres: The strands of time

Jonathan FayBMCB 625

June 14, 2007

Background• What is a telomere?• Why do we have them?• How do they get there?• What do they do?• Why do you care?• Gao et al. Nat Struct Mol Biol.

2007 Mar: 14(3):208-14

What is a telomere?

• 5-8 bp G-rich tandem repeats• Repetitive noncoding DNA

http://www.phoenixbiomolecular.com/regenerative_medicine.html

What is a telomere?What is a telomere?

Why do we have them?

• Replication problem– Lagging strand

synthesis • Unable to replicate

the 3’ ends faithfully• Loose chromosomal

DNA

http://www.uic.edu/classes/bios/bios100/lecturesf04am/ReplicationFork.gif

Evolutionary development of telomere

How do they get there?

• Telomerase

http://www.phoenixbiomolecular.com/regenerative_medicine.html

What do they do?Genetic Clock

telomeres are shortened each time a cell divides

Why do you care?• Telomeres gone bad….

Cancer and Age

DePinho, The age of cancer. Nature. 2000 Nov 9;408(6809):248-54.

Genetic Clock

Annu Rev Cell Dev Biol. 2006;22:531-57.

CrisisGenomic Instability

DePinho, The age of cancer. Nature. 2000 Nov 9;408(6809):248-54.

Genetic Clock

Annu Rev Cell Dev Biol. 2006;22:531-57.

immortalization

DePinho, The age of cancer. Nature. 2000 Nov 9;408(6809):248-54.

Summary

• Replication Problem– Evolutionary development of telomere

• Telomere 5-8 bp G-rich noncoding repetitive DNA

• Telomerase adds telomere to end of chromosome

• Telomere dysfunction can lead to cancer

What to they do?

• More than a genetic clock.• Protective cap

– Protect chromosomes form:• recombination, exonuclease degradation and

end-to-end fusion

– Distinguish telomeres from DNA ds breaks• That would hinder progression into G2 phase• Inappropriate recombination events

– Prevent Oncogenesis

Loss of telomere end protection leads to genome instability

Curr Opin Cell Biol. 2006 Jun;18(3):247-53.

What is the Cap?

• Nucleoprotein Complex– Number of different proteins that bind to

telomeres• ssDNA & dsDNA coat and protect the

telomere• Telomeric silencing• Structure protects ends

Structure: D-loop-T-loop

Cell Vol 97 419 199

G-Tetrad

G-quadruplex (G4)

http://www.nature.com/embor/journal/v7/n4/images/7400661-f1.jpgThe structure of telomeric DNA.Curr Opin Struct Biol. 2003 Jun;13(3):275-83

The Cap that is a lot of stuff!

Annu Rev Cell Dev Biol. 2006;22:531-57.

Diverse telomere-capping strategies

Cell biology. Telomere capping--one strand fits all.Science. 2001 May 11;292(5519):1075-6.

Budding Yeast Stn1 and Ten1 bind Cdc13

The structure of telomeric DNA.Curr Opin Struct Biol. 2003 Jun;13(3):275-83

Stn1 and Ten1 bind Cdc13

• Role of Cdc13– Cell cycle arrest mutant– Dual function protein

• Capping yeast telomeres• Recruit telomerase

• Unknown biochemical function of stn1 and ten1

Curr Opin Cell Biol. 2006 Jun;18(3):247-53.

OB fold – Bind oligonucleotides

or oligosaccarides– No significant amino

acid sequence similarity between OB fold proteins

– “Notorious for absence of primary sequence features that can be used to predict the domain.”

“Notable sequence conservation”

• OB-fold domain of Rpa2

• OB fold – Notorious for absence of

primary sequence

DNABindingDomain

“Notable sequence conservation”

• OB-fold domain of Rpa2• RPA (Replication protein

A)– Heterotrimer

• RPA2,RPA3,& RPA1– Core component of DNA

replication repair and recombination

– Binds ssDNA stabilizing unwound DNA and facilitates assembly of the complex through protein protein interactions

Does Stn1 bind DNA?

Domain Swap• Essential function

of RPA2• Restored by OB

fold of Stn1• Further evidence

N-term of Stn1 contains an OB fold– Perhaps an

evolutionary relationship between Stn1 and Rpa2

Does Ten1 bind DNA?

Rpa2,Rpa3 weak telomeric binding

Stn1 and Ten1 form a subcomplex

Domain Swap

N-terminal domains of Stn1 and Rpa2 are sufficient for viability

Summary

• Cdc13, Stn1 and Ten1 form RPA-like complex that binds telomeres

• Cdc13, Stn1 and Ten1 form RPA-like complex that binds telomeres

1

2

3

Rpa2 and Stn1 have similar biochemical activity •(Chimera)

Ten1 is like Rpa3 •It forms a complex with Stn1 or Rpa2 •It is the smallest subunit of RPA(like) complex

Rpa 2 & Rpa3 have specificity for telomeric DNA

Not just any ssDNA

Rpa can localize to chromosome ends

Competition??

Discussion Points• Reverse chimera Stn1-OBRPA2 didn’t work• Ten1 OB fold? Rosetta?• Oligomerization domain

– Cooperatively?

• G4 binding• Affinity? Is it too much to ask?

THEND

Sup 1

SUP 2

Sup 3

• Supplementary Figure 3. Comparison of the domain structure of subunits of the RPAand Cdc13-Stn1-Ten1 complexes.(a) Cdc13 and Rpa1 share a similar domain organization. Both proteins contain acentrally located OB-fold DNA binding domain (indicated in black), which binds withexceptionally high affinity to single-stranded DNA substrates of similar size (10-11 nt)1-5.As noted by Wuttke and colleagues, the extended conformation of single-stranded DNASupplemental Figure 3. (legend, continued).bound to the Cdc13 DBD is similar to that observed with RPA, but very distinct from thatassumed by single-stranded DNA in complex with O. nova TEBP6. Furthermore, the Pot1protein (which exhibits weak sequence similarity with the ! subunit of the O. nova TEBPcomplex) has a different domain structure from that of Cdc13 and Rpa1; most notably,high affinity binding is mediated through two OB-folds located in the extreme N-terminus ofthe Pot1 protein7,8. Rpa1 contains an additional OB-fold in its C-terminal region9,10(indicated by a grey box), and an OB-fold has also been detected in the C-terminal domainof Cdc1311 (grey box), using a sensitive sequence profile comparison program. Finally,the N-terminal regions of Cdc13 and Rpa1 each serve as protein-protein interactionmodules. Cdc13 interacts with the Est1 subunit of telomerase, mediated through a 15 kDadomain located at aa 211 to 331 of Cdc1312,13. Rpa1 also has a well-characterized Nterminal120 amino acid domain that interacts with several different protein complexes,including the p53 tumor suppressor protein14.(b) Both Stn1 and Rpa2 contain a single OB-fold, in the N-terminal half of the protein,which performs essential roles in each protein. The OB-fold domain shown in Fig. 1 isindicated by a black box, and the boundaries of the essential domain defined by theexperiment in Fig. 5c are bracketed.(c) The smallest subunit of the RPA complex, Rpa3, is folded into a single OB-folddomain15, indicated by a black box. Using the bioinformatics techniques that uncoveredsimilarities between Stn1 and Rpa2, we were not able to detect comparable sequenceidentity between Rpa3 and Ten1, and therefore we cannot conclude whether Ten1contains an OB-fold domain or not. This may be a reflection of the fact that both proteinshave diverged rapidly at the primary sequence level, as revealed by the alignments ofRpa3 and Ten1 sequences from fungal genomes. Although the Rpa3 alignment shownhere, composed from a collection of fungal Rpa3 proteins, reveals a modest degree ofsequence conservation, we nevertheless were unable to place more distantly related Rpa3proteins, such as the human homolog, on this alignment. The Ten1 protein family appearsto be even more divergent; for example, we were only able to identify the A. gossypii Ten1sequence based on its syntenic position in the genome, rather than by a BLAST search.We have so far been unable to recover additional Ten1 proteins, even from other fungalgenomes, further suggesting rapid sequence divergence within the Ten1 family.