Identify forces responsible for chromosome movement Determine the repertoire of chromosome movement in segregation, repair and recombination What is the cellular response, including chromosome reorganization to DNA damage?Is chromosome movement integral for DNA repair?

479data

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4213:54.614:39.256:57.2194.552345.08791193.7489910.57755.5480599844

2501data

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103:09:11 PM0:04:29109.6402614.996109.9080415.12980.2993469031

113:09:41 PM0:05:00119.3724715.1298119.7741515.12980.40168

123:10:11 PM0:05:30129.5063615.1298129.5063614.9960.1338

133:10:41 PM0:06:00139.1046914.996139.5063614.86210.4234005183

143:11:11 PM0:06:29149.1046914.8621149.3724714.72820.2993916138

153:11:41 PM0:06:59158.836915.1298158.9707914.9960.189285425

163:12:11 PM0:07:29168.9707915.1298169.2385815.12980.26779

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183:13:11 PM0:08:29189.1046914.996189.2385814.9960.13389

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203:14:11 PM0:09:30209.5063614.5943209.9080414.72820.4234100051

213:14:41 PM0:10:00219.9080414.5943219.9080414.59430

223:15:11 PM0:10:292210.175814.46042210.175814.46040

233:15:41 PM0:10:59239.9080414.46042310.041914.59430.1893349138

243:16:11 PM0:11:29249.9080414.5943249.9080414.46040.1339

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273:17:41 PM0:13:00279.9080414.46042710.309714.46040.40166

283:18:11 PM0:13:302810.175814.46042810.443614.59430.2994095022

293:18:41 PM0:13:592910.175814.46042910.309714.59430.189363196

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313:19:41 PM0:14:593110.443614.59433110.577514.59430.1339

323:20:11 PM0:15:303210.577514.72823210.845314.86210.2994095022

333:20:41 PM0:15:593310.577514.72823310.845314.86210.2994095022

343:21:11 PM0:16:303410.711414.72823410.845314.72820.1339

353:21:41 PM0:16:593510.443614.72823510.577514.72820.1339

363:22:11 PM0:17:303610.711414.46043610.845314.59430.189363196

373:22:41 PM0:17:593710.175814.59433710.309714.59430.1339

383:23:11 PM0:18:303810.041914.59433810.041914.59430

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403:24:11 PM0:19:304010.041914.32654010.041914.46040.1339

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433:25:41 PM0:21:004310.309714.46044310.309714.59430.1339

443:26:11 PM0:21:294410.175814.46044410.443614.46040.2678

453:26:41 PM0:21:594510.041914.46044510.309714.46040.2678

463:27:13 PM0:22:314610.309714.59434610.443614.59430.1339

473:29:12 PM0:24:31479.6402614.996479.6402615.12980.1338

483:31:13 PM0:26:31489.1046915.2637489.1046915.12980.1339

493:33:12 PM0:28:31499.5063615.1298499.5063615.26370.1339

503:35:13 PM0:30:31509.7741514.32655010.175814.19260.4233815448

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533:41:13 PM0:36:31539.6402614.5943539.7741514.46040.1893561251

543:43:13 PM0:38:31548.7030114.8621549.9080415.79931.5265782459

553:45:13 PM0:40:31557.6318714.05875510.845315.53153.5348652315

563:47:13 PM0:42:31567.2301914.05875611.648615.39764.6168171004

573:49:13 PM0:44:31576.5607314.46045711.916414.59435.3573435916

583:51:13 PM0:46:31586.2929414.59435811.782514.46045.4911927851

593:53:13 PM0:48:31596.5607315.12985912.184214.59435.6489091948

603:55:13 PM0:50:31606.2929414.59436012.853714.72826.5621262551

613:57:13 PM0:52:31615.2218114.9966111.648614.86216.4281847293

623:59:13 PM0:54:31625.4895915.12986212.45214.59436.9829730959

634:01:13 PM0:56:31635.2218113.92486312.45214.9967.3091119075

644:03:13 PM0:58:31644.5523414.05876412.318114.86217.8072069229

654:05:13 PM1:00:31654.8201314.32656512.585914.32657.76577

664:07:12 PM1:02:31664.6862414.19266613.121514.46048.4395099448

674:09:13 PM1:04:31675.0879113.79096712.987615.12988.0123501737

684:11:13 PM1:06:31684.8201313.79096813.121515.26378.4310072777

694:13:13 PM1:08:31694.6862413.92486912.987615.66548.4818786958

704:15:13 PM1:10:31704.8201313.6577012.853715.66548.2808162342

714:17:12 PM1:12:31714.9540213.79097112.318116.06717.7078376142

724:19:12 PM1:14:31725.0879113.52317212.853716.2018.2145384979

734:21:12 PM1:16:31734.9540213.52317312.45215.93327.8758038377

Comparison Chart

0.133890.06695

0.48271121010.0748467854

0.18929249850.033475

0.13380.0334725

0.803350.2840288841

0.42337839150.5490018604

0.13390.7984616988

0.13390.9707183621

0.29939161380.9952264618

0.29934690310.9730239484

0.401681.0246225269

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CEN spot separation

25Kb from CEN3 spot separation

Elapsed Time (hrs:min:sec)

Spot Separation Distance (um)

Comparison Timecourse of Chromosome Markers 1Kb and 25Kb from CEN3

Yeast chromosomes oscillate from pole to pole prior to anaphase onset.

Centromeres separate prior to the chromosome armsRate of separation of chromosome arms exceeds rate of centromere separation Mechanisms of cohesion and separation are likely to differ along the chromatin fiber

Centromeres on the same chromatid attach to opposite poles: Chromosome BreakageCentromeres on the same chromatid attach to same poles:NO Chromosome Breakage

865bp1100bpRAD+rad52- or rad1-Repair mutant Chromosome rearrangements visualized in time course from galactose to glucoseGalGalGluGlu

Single strand excisionRepair via Single strand annealingRevealed by 1100 bp repair product

Activate Dicentric Chromosome mid-anaphase delay follows breakDoes mid-anaphase delay precede break ?or

Mid-anaphase pause is dependent upon RAD9 checkpoint geneMid-anaphase pause is not dependent upon the spindle assembly checkpoint genes, MAD2 or BUB1RAD9 recognizes DNA damage and/or chromatin alterationsDoes RAD9 mobilize chromosomes/repair machinery in response to DNA damage

Viability of cells upon activation of the dicentric chromosome Repair mutants (rad1, rad52 and Yku70), rad9 checkpointand mt based motility mutants (dynein, kip3, kar9)

Chart1

60

70

1

20

23

21

28

15

Viability %

Sheet1

StrainViability %

Wildtype60

rad970

rad521

rad120

yku7023

dynein21

kip328

kar915

kip3,kar979

Sheet1

Viability %

Sheet2

Viability %

Sheet3

Cytoplasmic microtubule-based motor proteins contribute to the fidelity of chromosome repairHow do repair mutants effect chromosome organizationThe mid-anaphase pause is longer in the absence of cytoplasmic dynein or kip3Mt based motor proteins effect the DNA damage response

Ku- Non-homologous end-joining

Ku70, Ku80, and DNA-PKcs associate to form DNA-PK

Featherstone, C., and Jackson, S. Mutat Res. 1999 May 14;434(1):3-15. Review.

Two subunits bind tightly to each other, then form a tetramer (or dimer of dimers) when bound to breakage site.

After binding, recruits 470 kDa DNA-dependent protein kinase catalytic subunit.

2x Ku702xKu802xDNA-PKcs= DNA-PK

DNA-PK phosphorylates target proteins such as RNA pol II

Physiologicalfunctions of KuKu70, Ku80 knockouts in mice have similar phenotype to SCIDV(D)J defects arrest lymphocyte developmentKu70, Ku80 -/- mice are runts compared to +/- littermatesNumber of cell divisions in development limited by impaired ability to repair endogenously generated DNA damageKu-deficient cells might take longer to repair this damageKu80 -/- dams fail to nurture their pups

Yeast Ku intelomere maintenanceDisruption of yKu70p and yKu80p genes affect telomeric silencing, telomere length maintenance and viability of cells containing dicentric chromosomesinactivate Ku, lose telomeric silencinginactivate Ku, shorten telomeresinactivate Ku, decrease viability of cells containing dicentric plasmids

Ku clusters yeast telomeres to peripheral sites in nucleusIn diploids, telomeres usually found in 6-7 clusters around nuclear peripheryIn Ku subunit mutants, more clusters in random locationsFeatherstone, C., and Jackson, S. Mutat Res. 1999 May 14;434(1):3-15. Review.

Activation of dicentric chromosome in Ku mutants results in chromosome decondensation

Chromosome decondenses in stepwise fashion

Dicentric chromosome stretching in metaphase in a stepwise fashion and recoil upon spindle disassembly

Regularity of spacing upon chromatin decompaction

Chromosome breakage and retraction to spindle poles

Sir2p binds to stretched lacO DNAChromatin stretching is recognized in vivo Chromatin can act as an in vivo tensiometerMAT

RAD9+rad9-Rad9 is required for chromosome unfolding in response to activation of the dicentric chromosome

Laboratory ProjectsElaine Yeh- Asymmetric protein determinantsDale Beach- RNA localizationPaul Maddox-Mt polarity and dynamics

THANKS !!!

We already know that (Point 1).

Point 2. 40-60% size of littermate controls.

Ku80 -/- females are unable to sustain their pups, which die within a few days unless they are nursed by a foster mother. This finding was from a Letter to Nature, and the authors did not expound on why this is so.When the genes that encode either Yku70p or yKu80p are disrupted, not only NHEJ but also telomeric silencing and telomere length maintenance are significantly perturbed. Telomeric silencing (aka telomere position effect) occurs when a gene is engineered into the telomeric region of a yeast chromosome. In these end regions, the chromatin is in a unique condensed structure that doesnt permit access by the transcriptional apparatus and therefore the engineered gene is silent. If this heterochromatin-like state is disrupted, telomeric silencing is relieved and genes can be expressed. When Ku is inactivated, this occurs as well as the loss of about 65% of telomere length.

How is this possible? Perhaps Ku protects telomeric DNA ends from degradation. Point 2.At another level, Ku appears to be involved in the clustering of yeast telomeres at peripheral sites in the nucleus. In wild-type diploid yeast cells, the 63 telomeres are usually found in six or seven clusters around the nuclear periphery, while cells mutant in either Ku subunit gene have around nine clusters that seem to be located more randomly throughout the nucleus. These results suggest Ku is somehow involved in clustering telomeres and tethering them at sites in the nuclear periphery.