Cdk1 Targets Srs2 to Complete Synthesis-Dependent Strand Annealing and to Promote Recombinational Repair

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Cdk1 kinase phosphorylates budding yeast Srs2, a member of UvrD protein family, displays both DNA translocation and DNA unwinding activities in vitro. Srs2 prevents homologous recombination by dismantling Rad51 filaments and is also required for double-strand break (DSB) repair. Here we examine the biological significance of Cdk1-dependent phosphorylation of Srs2, using mutants that constitutively express the phosphorylated or unphosphorylated protein isoforms. We found that Cdk1 targets Srs2 to repair DSB and, in particular, to complete synthesis-dependent strand annealing, likely controlling the disassembly of a D-loop intermediate. Cdk1-dependent phosphorylation controls turnover of Srs2 at the invading strand; and, in absence of this modification, the turnover of Rad51 is not affected. Further analysis of the recombination phenotypes of the srs2 phospho-mutants showed that Srs2 phosphorylation is not required for the removal of toxic Rad51 nucleofilaments, although it is essential for cell survival, when DNA breaks are channeled into homologous recombinational repair. Cdk1-targeted Srs2 displays a PCNA–independent role and appears to have an attenuated ability to inhibit recombination. Finally, the recombination defects of unphosphorylatable Srs2 are primarily due to unscheduled accumulation of the Srs2 protein in a sumoylated form. Thus, the Srs2 anti-recombination function in removing toxic Rad51 filaments is genetically separable from its role in promoting recombinational repair, which depends exclusively on Cdk1-dependent phosphorylation. We suggest that Cdk1 kinase counteracts unscheduled sumoylation of Srs2 and targets Srs2 to dismantle specific DNA structures, such as the D-loops, in a helicase-dependent manner during homologous recombinational repair.

Vyšlo v časopise: Cdk1 Targets Srs2 to Complete Synthesis-Dependent Strand Annealing and to Promote Recombinational Repair. PLoS Genet 6(2): e32767. doi:10.1371/journal.pgen.1000858
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1000858

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Zdroje

1. Keen-KimD

NooraieF

RaoPN

2008 Cytogenetic biomarkers for human cancer. Front Biosci 13 5928 5949

2. PaquesF

HaberJE

1999 Multiple pathways of recombination induced by double-strand breaks in Saccharomyces cerevisiae. Microbiol Mol Biol Rev 63 349 404

3. KroghBO

SymingtonLS

2004 Recombination proteins in yeast. Annu Rev Genet 38 233 271

4. San FilippoJ

SungP

KleinH

2008 Mechanism of eukaryotic homologous recombination. Annu Rev Biochem 77 229 257

5. LiuY

WestSC

2004 Happy Hollidays: 40th anniversary of the Holliday junction. Nat Rev Mol Cell Biol 5 937 944

6. JainS

SugawaraN

LydeardJ

VazeM

Tanguy Le GacN

2009 A recombination execution checkpoint regulates the choice of homologous recombination pathway during DNA double-strand break repair. Genes Dev 23 291 303

7. AgmonN

PurS

LiefshitzB

KupiecM

2009 Analysis of repair mechanism choice during homologous recombination. Nucleic Acids Res 37 5081 5092

8. NagaiS

DubranaK

Tsai-PflugfelderM

DavidsonMB

RobertsTM

2008 Functional targeting of DNA damage to a nuclear pore-associated SUMO-dependent ubiquitin ligase. Science 322 597 602

9. OzaP

JaspersenSL

MieleA

DekkerJ

PetersonCL

2009 Mechanisms that regulate localization of a DNA double-strand break to the nuclear periphery. Genes Dev 23 912 927

10. ConradMN

LeeCY

WilkersonJL

DresserME

2007 MPS3 mediates meiotic bouquet formation in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 104 8863 8868

11. MimitouEP

SymingtonLS

2009 Nucleases and helicases take center stage in homologous recombination. Trends Biochem Sci 34 264 272

12. SzostakJW

Orr-WeaverTL

RothsteinRJ

StahlFW

1983 The double-strand-break repair model for recombination. Cell 33 25 35

13. GangloffS

SoustelleC

FabreF

2000 Homologous recombination is responsible for cell death in the absence of the Sgs1 and Srs2 helicases. Nat Genet 25 192 194

14. KleinHL

2001 Mutations in recombinational repair and in checkpoint control genes suppress the lethal combination of srs2Delta with other DNA repair genes in Saccharomyces cerevisiae. Genetics 157 557 565

15. BurgessRC

LisbyM

AltmannovaV

KrejciL

SungP

2009 Localization of recombination proteins and Srs2 reveals anti-recombinase function in vivo. J Cell Biol 185 969 981

16. AboussekhraA

ChanetR

AdjiriA

FabreF

1992 Semidominant suppressors of Srs2 helicase mutations of Saccharomyces cerevisiae map in the RAD51 gene, whose sequence predicts a protein with similarities to procaryotic RecA proteins. Mol Cell Biol 12 3224 3234

17. MalikPS

SymingtonLS

2008 Rad51 gain-of-function mutants that exhibit high affinity DNA binding cause DNA damage sensitivity in the absence of Srs2. Nucleic Acids Res 36 6504 6510

18. VeauteX

JeussetJ

SoustelleC

KowalczykowskiSC

Le CamE

2003 The Srs2 helicase prevents recombination by disrupting Rad51 nucleoprotein filaments. Nature 423 309 312

19. KrejciL

Van KomenS

LiY

VillemainJ

ReddyMS

2003 DNA helicase Srs2 disrupts the Rad51 presynaptic filament. Nature 423 305 309

20. PapouliE

ChenS

DaviesAA

HuttnerD

KrejciL

2005 Crosstalk between SUMO and ubiquitin on PCNA is mediated by recruitment of the helicase Srs2p. Mol Cell 19 123 133

21. PfanderB

MoldovanGL

SacherM

HoegeC

JentschS

2005 SUMO-modified PCNA recruits Srs2 to prevent recombination during S phase. Nature 436 428 433

22. RongL

KleinHL

1993 Purification and characterization of the SRS2 DNA helicase of the yeast Saccharomyces cerevisiae. J Biol Chem 268 1252 1259

23. DupaigneP

Le BretonC

FabreF

GangloffS

Le CamE

2008 The Srs2 helicase activity is stimulated by Rad51 filaments on dsDNA: implications for crossover incidence during mitotic recombination. Mol Cell 29 243 254

24. BlanckS

KobbeD

HartungF

FenglerK

FockeM

2009 A SRS2 homolog from Arabidopsis thaliana disrupts recombinogenic DNA intermediates and facilitates single strand annealing. Nucleic Acids Res

25. IraG

MalkovaA

LiberiG

FoianiM

HaberJE

2003 Srs2 and Sgs1-Top3 suppress crossovers during double-strand break repair in yeast. Cell 115 401 411

26. RobertT

DervinsD

FabreF

GangloffS

2006 Mrc1 and Srs2 are major actors in the regulation of spontaneous crossover. Embo J 25 2837 2846

27. Welz-VoegeleC

Jinks-RobertsonS

2008 Sequence divergence impedes crossover more than noncrossover events during mitotic gap repair in yeast. Genetics 179 1251 1262

28. AylonY

LiefshitzB

Bitan-BaninG

KupiecM

2003 Molecular dissection of mitotic recombination in the yeast Saccharomyces cerevisiae. Mol Cell Biol 23 1403 1417

29. VazeMB

PellicioliA

LeeSE

IraG

LiberiG

2002 Recovery from checkpoint-mediated arrest after repair of a double-strand break requires Srs2 helicase. Mol Cell 10 373 385

30. LiberiG

ChioloI

PellicioliA

LopesM

PlevaniP

2000 Srs2 DNA helicase is involved in checkpoint response and its regulation requires a functional Mec1-dependent pathway and Cdk1 activity. Embo J 19 5027 5038

31. UbersaxJA

WoodburyEL

QuangPN

ParazM

BlethrowJD

2003 Targets of the cyclin-dependent kinase Cdk1. Nature 425 859 864

32. WohlboldL

FisherRP

2009 Behind the wheel and under the hood: functions of cyclin-dependent kinases in response to DNA damage. DNA Repair (Amst) 8 1018 1024

33. IraG

PellicioliA

BalijjaA

WangX

FioraniS

2004 DNA end resection, homologous recombination and DNA damage checkpoint activation require CDK1. Nature 431 1011 1017

34. AylonY

LiefshitzB

KupiecM

2004 The CDK regulates repair of double-strand breaks by homologous recombination during the cell cycle. Embo J 23 4868 4875

35. HuertasP

Cortes-LedesmaF

SartoriAA

AguileraA

JacksonSP

2008 CDK targets Sae2 to control DNA-end resection and homologous recombination. Nature 455 689 692

36. LeeJY

YangW

2006 UvrD helicase unwinds DNA one base pair at a time by a two-part power stroke. Cell 127 1349 1360

37. AntonyE

TomkoEJ

XiaoQ

KrejciL

LohmanTM

2009 Srs2 disassembles Rad51 filaments by a protein-protein interaction triggering ATP turnover and dissociation of Rad51 from DNA. Mol Cell 35 105 115

38. ColavitoS

Macris-KissM

SeongC

GleesonO

GreeneEC

2009 Functional significance of the Rad51-Srs2 complex in Rad51 presynaptic filament disruption. Nucleic Acids Res

39. ChioloI

CarotenutoW

MaffiolettiG

PetriniJH

FoianiM

2005 Srs2 and Sgs1 DNA helicases associate with Mre11 in different subcomplexes following checkpoint activation and CDK1-mediated Srs2 phosphorylation. Mol Cell Biol 25 5738 5751

40. Le BretonC

DupaigneP

RobertT

Le CamE

GangloffS

2008 Srs2 removes deadly recombination intermediates independently of its interaction with SUMO-modified PCNA. Nucleic Acids Res 36 4964 4974

41. DebrauwereH

LoeilletS

LinW

LopesJ

NicolasA

2001 Links between replication and recombination in Saccharomyces cerevisiae: a hypersensitive requirement for homologous recombination in the absence of Rad27 activity. Proc Natl Acad Sci U S A 98 8263 8269

42. SymingtonLS

1998 Homologous recombination is required for the viability of rad27 mutants. Nucleic Acids Res 26 5589 5595

43. AboussekhraA

ChanetR

ZgagaZ

Cassier-ChauvatC

HeudeM

1989 RADH, a gene of Saccharomyces cerevisiae encoding a putative DNA helicase involved in DNA repair. Characteristics of radH mutants and sequence of the gene. Nucleic Acids Res 17 7211 7219

44. PalladinoF

KleinHL

1992 Analysis of mitotic and meiotic defects in Saccharomyces cerevisiae SRS2 DNA helicase mutants. Genetics 132 23 37

45. HarrisonJC

HaberJE

2006 Surviving the breakup: the DNA damage checkpoint. Annu Rev Genet 40 209 235

46. PradoF

AguileraA

2003 Control of cross-over by single-strand DNA resection. Trends Genet 19 428 431

47. CarterSD

VigasovaD

ChenJ

ChovanecM

AstromSU

2009 Nej1 recruits the Srs2 helicase to DNA double-strand breaks and supports repair by a single-strand annealing-like mechanism. Proc Natl Acad Sci U S A

48. LiuY

KaoHI

BambaraRA

2004 Flap endonuclease 1: a central component of DNA metabolism. Annu Rev Biochem 73 589 615

49. LuccaC

VanoliF

Cotta-RamusinoC

PellicioliA

LiberiG

2004 Checkpoint-mediated control of replisome-fork association and signalling in response to replication pausing. Oncogene 23 1206 1213

50. LivakKJ

SchmittgenTD