The SUMO Isopeptidase Ulp2p Is Required to Prevent Recombination-Induced Chromosome Segregation Lethality following DNA Replication Stress
SUMO conjugation is a key regulator of the cellular response to DNA replication stress, acting in part to control recombination at stalled DNA replication forks. Here we examine recombination-related phenotypes in yeast mutants defective for the SUMO de-conjugating/chain-editing enzyme Ulp2p. We find that spontaneous recombination is elevated in ulp2 strains and that recombination DNA repair is essential for ulp2 survival. In contrast to other SUMO pathway mutants, however, the frequency of spontaneous chromosome rearrangements is markedly reduced in ulp2 strains, and some types of rearrangements arising through recombination can apparently not be tolerated. In investigating the basis for this, we find DNA repair foci do not disassemble in ulp2 cells during recovery from the replication fork-blocking drug methyl methanesulfonate (MMS), corresponding with an accumulation of X-shaped recombination intermediates. ulp2 cells satisfy the DNA damage checkpoint during MMS recovery and commit to chromosome segregation with similar kinetics to wild-type cells. However, sister chromatids fail to disjoin, resulting in abortive chromosome segregation and cell lethality. This chromosome segregation defect can be rescued by overproducing the anti-recombinase Srs2p, indicating that recombination plays an underlying causal role in blocking chromatid separation. Overall, our results are consistent with a role for Ulp2p in preventing the formation of DNA lesions that must be repaired through recombination. At the same time, Ulp2p is also required to either suppress or resolve recombination-induced attachments between sister chromatids. These opposing defects may synergize to greatly increase the toxicity of DNA replication stress.
Vyšlo v časopise:
The SUMO Isopeptidase Ulp2p Is Required to Prevent Recombination-Induced Chromosome Segregation Lethality following DNA Replication Stress. PLoS Genet 7(3): e32767. doi:10.1371/journal.pgen.1001355
Kategorie:
Research Article
prolekare.web.journal.doi_sk:
https://doi.org/10.1371/journal.pgen.1001355
Souhrn
SUMO conjugation is a key regulator of the cellular response to DNA replication stress, acting in part to control recombination at stalled DNA replication forks. Here we examine recombination-related phenotypes in yeast mutants defective for the SUMO de-conjugating/chain-editing enzyme Ulp2p. We find that spontaneous recombination is elevated in ulp2 strains and that recombination DNA repair is essential for ulp2 survival. In contrast to other SUMO pathway mutants, however, the frequency of spontaneous chromosome rearrangements is markedly reduced in ulp2 strains, and some types of rearrangements arising through recombination can apparently not be tolerated. In investigating the basis for this, we find DNA repair foci do not disassemble in ulp2 cells during recovery from the replication fork-blocking drug methyl methanesulfonate (MMS), corresponding with an accumulation of X-shaped recombination intermediates. ulp2 cells satisfy the DNA damage checkpoint during MMS recovery and commit to chromosome segregation with similar kinetics to wild-type cells. However, sister chromatids fail to disjoin, resulting in abortive chromosome segregation and cell lethality. This chromosome segregation defect can be rescued by overproducing the anti-recombinase Srs2p, indicating that recombination plays an underlying causal role in blocking chromatid separation. Overall, our results are consistent with a role for Ulp2p in preventing the formation of DNA lesions that must be repaired through recombination. At the same time, Ulp2p is also required to either suppress or resolve recombination-induced attachments between sister chromatids. These opposing defects may synergize to greatly increase the toxicity of DNA replication stress.
Zdroje
1. BroomfieldS
HryciwT
XiaoW
2001 DNA postreplication repair and mutagenesis in Saccharomyces cerevisiae. Mutat Res 486 167 184
2. San FilippoJ
SungP
KleinH
2008 Mechanism of eukaryotic homologous recombination. Annu Rev Biochem 77 229 257
3. BranzeiD
FoianiM
2010 Maintaining genome stability at the replication fork. Nat Rev Mol Cell Biol 11 208 219
4. JohnsonES
2004 Protein modification by SUMO. Annu Rev Biochem 73 355 382
5. HoegeC
PfanderB
MoldovanGL
PyrowolakisG
JentschS
2002 RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO. Nature 419 135 141
6. PfanderB
MoldovanGL
SacherM
HoegeC
JentschS
2005 SUMO-modified PCNA recruits Srs2 to prevent recombination during S phase. Nature 436 428 433
7. 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
8. KrejciL
Van KomenS
LiY
VillemainJ
ReddyMS
2003 DNA helicase Srs2 disrupts the Rad51 presynaptic filament. Nature 423 305 309
9. VeauteX
JeussetJ
SoustelleC
KowalczykowskiSC
Le CamE
2003 The Srs2 helicase prevents recombination by disrupting Rad51 nucleoprotein filaments. Nature 423 309 312
10. 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
11. StelterP
UlrichHD
2003 Control of spontaneous and damage-induced mutagenesis by SUMO and ubiquitin conjugation. Nature 425 188 191
12. HaracskaL
Torres-RamosCA
JohnsonRE
PrakashS
PrakashL
2004 Opposing effects of ubiquitin conjugation and SUMO modification of PCNA on replicational bypass of DNA lesions in Saccharomyces cerevisiae. Mol Cell Biol 24 4267 4274
13. KannouchePL
WingJ
LehmannAR
2004 Interaction of human DNA polymerase eta with monoubiquitinated PCNA: a possible mechanism for the polymerase switch in response to DNA damage. Mol Cell 14 491 500
14. MaedaD
SekiM
OnodaF
BranzeiD
KawabeY
2004 Ubc9 is required for damage-tolerance and damage-induced interchromosomal homologous recombination in S. cerevisiae. DNA Repair (Amst) 3 335 341
15. AndrewsEA
PalecekJ
SergeantJ
TaylorE
LehmannAR
2005 Nse2, a component of the Smc5-6 complex, is a SUMO ligase required for the response to DNA damage. Mol Cell Biol 25 185 196
16. ZhaoX
BlobelG
2005 A SUMO ligase is part of a nuclear multiprotein complex that affects DNA repair and chromosomal organization. Proc Natl Acad Sci U S A 102 4777 4782
17. BranzeiD
SollierJ
LiberiG
ZhaoX
MaedaD
2006 Ubc9- and mms21-mediated sumoylation counteracts recombinogenic events at damaged replication forks. Cell 127 509 522
18. van WaardenburgRC
DudaDM
LancasterCS
SchulmanBA
BjornstiMA
2006 Distinct functional domains of Ubc9 dictate cell survival and resistance to genotoxic stress. Mol Cell Biol 26 4958 4969
19. BranzeiD
VanoliF
FoianiM
2008 SUMOylation regulates Rad18-mediated template switch. Nature 456 915 920
20. OnodaF
TakedaM
SekiM
MaedaD
TajimaJ
2004 SMC6 is required for MMS-induced interchromosomal and sister chromatid recombinations in Saccharomyces cerevisiae. DNA Repair (Amst) 3 429 439
21. Torres-RosellJ
MachinF
FarmerS
JarmuzA
EydmannT
2005 SMC5 and SMC6 genes are required for the segregation of repetitive chromosome regions. Nat Cell Biol 7 412 419
22. AmpatzidouE
IrmischA
O'ConnellMJ
MurrayJM
2006 Smc5/6 is required for repair at collapsed replication forks. Mol Cell Biol 26 9387 9401
23. SollierJ
DriscollR
CastellucciF
FoianiM
JacksonSP
2009 The Saccharomyces cerevisiae Esc2 and Smc5-6 proteins promote sister chromatid junction-mediated intra-S repair. Mol Biol Cell 20 1671 1682
24. KarowJK
ConstantinouA
LiJL
WestSC
HicksonID
2000 The Bloom's syndrome gene product promotes branch migration of holliday junctions. Proc Natl Acad Sci U S A 97 6504 6508
25. LiberiG
MaffiolettiG
LuccaC
ChioloI
BaryshnikovaA
2005 Rad51-dependent DNA structures accumulate at damaged replication forks in sgs1 mutants defective in the yeast ortholog of BLM RecQ helicase. Genes Dev 19 339 350
26. PlankJL
WuJ
HsiehTS
2006 Topoisomerase IIIalpha and Bloom's helicase can resolve a mobile double Holliday junction substrate through convergent branch migration. Proc Natl Acad Sci U S A 103 11118 11123
27. MankouriHW
NgoHP
HicksonID
2007 Shu proteins promote the formation of homologous recombination intermediates that are processed by Sgs1-Rmi1-Top3. Mol Biol Cell 18 4062 4073
28. LiSJ
HochstrasserM
1999 A new protease required for cell-cycle progression in yeast. Nature 398 246 251
29. LiSJ
HochstrasserM
2000 The yeast ULP2 (SMT4) gene encodes a novel protease specific for the ubiquitin-like Smt3 protein. Mol Cell Biol 20 2367 2377
30. SchwienhorstI
JohnsonES
DohmenRJ
2000 SUMO conjugation and deconjugation. Mol Gen Genet 263 771 786
31. LiSJ
HochstrasserM
2003 The Ulp1 SUMO isopeptidase: distinct domains required for viability, nuclear envelope localization, and substrate specificity. J Cell Biol 160 1069 1081
32. PanseVG
KusterB
GerstbergerT
HurtE
2003 Unconventional tethering of Ulp1 to the transport channel of the nuclear pore complex by karyopherins. Nat Cell Biol 5 21 27
33. KroetzMB
SuD
HochstrasserM
2009 Essential role of nuclear localization for yeast Ulp2 SUMO protease function. Mol Biol Cell 20 2196 2206
34. MeluhPB
KoshlandD
1995 Evidence that the MIF2 gene of Saccharomyces cerevisiae encodes a centromere protein with homology to the mammalian centromere protein CENP-C. Mol Biol Cell 6 793 807
35. StrunnikovAV
AravindL
KooninEV
2001 Saccharomyces cerevisiae SMT4 encodes an evolutionarily conserved protease with a role in chromosome condensation regulation. Genetics 158 95 107
36. BachantJ
AlcasabasA
BlatY
KlecknerN
ElledgeSJ
2002 The SUMO-1 isopeptidase Smt4 is linked to centromeric cohesion through SUMO-1 modification of DNA topoisomerase II. Mol Cell 9 1169 1182
37. SteadK
AguilarC
HartmanT
DrexelM
MeluhP
2003 Pds5p regulates the maintenance of sister chromatid cohesion and is sumoylated to promote the dissolution of cohesion. J Cell Biol 163 729 741
38. D'AmoursD
StegmeierF
AmonA
2004 Cdc14 and condensin control the dissolution of cohesin-independent chromosome linkages at repeated DNA. Cell 117 455 469
39. BachantJ
JessenSR
KavanaughSE
FieldingCS
2005 The yeast S phase checkpoint enables replicating chromosomes to bi-orient and restrain spindle extension during S phase distress. J Cell Biol 168 999 1012
40. TakahashiY
Yong-GonzalezV
KikuchiY
StrunnikovA
2006 SIZ1/SIZ2 control of chromosome transmission fidelity is mediated by the sumoylation of topoisomerase II. Genetics 172 783 794
41. SchwartzDC
FelberbaumR
HochstrasserM
2007 The Ulp2 SUMO protease is required for cell division following termination of the DNA damage checkpoint. Mol Cell Biol 27 6948 6961
42. BaldwinML
JuliusJA
TangX
WangY
BachantJ
2009 The yeast SUMO isopeptidase Smt4/Ulp2 and the polo kinase Cdc5 act in an opposing fashion to regulate sumoylation in mitosis and cohesion at centromeres. Cell Cycle 8 3406 3419
43. BylebylGR
BelichenkoI
JohnsonES
2003 The SUMO isopeptidase Ulp2 prevents accumulation of SUMO chains in yeast. J Biol Chem 278 44113 44120
44. UzunovaK
GottscheK
MitevaM
WeisshaarSR
GlanemannC
2007 Ubiquitin-dependent proteolytic control of SUMO conjugates. J Biol Chem 282 34167 34175
45. TathamMH
GeoffroyMC
ShenL
PlechanovovaA
HattersleyN
2008 RNF4 is a poly-SUMO-specific E3 ubiquitin ligase required for arsenic-induced PML degradation. Nat Cell Biol 10 538 546
46. MullenJR
BrillSJ
2008 Activation of the Slx5-Slx8 ubiquitin ligase by poly-small ubiquitin-like modifier conjugates. J Biol Chem 283 19912 19921
47. SoustelleC
VernisL
FreonK
Reynaud-AngelinA
ChanetR
2004 A new Saccharomyces cerevisiae strain with a mutant Smt3-deconjugating Ulp1 protein is affected in DNA replication and requires Srs2 and homologous recombination for its viability. Mol Cell Biol 24 5130 5143
48. FelberbaumR
HochstrasserM
2008 Ulp2 and the DNA damage response: desumoylation enables safe passage through mitosis. Cell Cycle 7 52 56
49. LisbyM
RothsteinR
MortensenUH
2001 Rad52 forms DNA repair and recombination centers during S phase. Proc Natl Acad Sci U S A 98 8276 8282
50. MainesS
NegrittoMC
WuX
MantheyGM
BailisAM
1998 Novel mutations in the RAD3 and SSL1 genes perturb genome stability by stimulating recombination between short repeats in Saccharomyces cerevisiae. Genetics 150 963 976
51. LambertS
WatsonA
SheedyDM
MartinB
CarrAM
2005 Gross chromosomal rearrangements and elevated recombination at an inducible site-specific replication fork barrier. Cell 121 689 702
52. LemoineFJ
DegtyarevaNP
KokoskaRJ
PetesTD
2008 Reduced levels of DNA polymerase delta induce chromosome fragile site instability in yeast. Mol Cell Biol 28 5359 5368
53. HuangD
KoshlandD
2003 Chromosome integrity in Saccharomyces cerevisiae: the interplay of DNA replication initiation factors, elongation factors, and origins. Genes Dev 17 1741 1754
54. MannC
DavisRW
1983 Instability of dicentric plasmids in yeast. Proc Natl Acad Sci U S A 80 228 232
55. KoshlandD
RutledgeL
Fitzgerald-HayesM
HartwellLH
1987 A genetic analysis of dicentric minichromosomes in Saccharomyces cerevisiae. Cell 48 801 812
56. BarlowJH
RothsteinR
2009 Rad52 recruitment is DNA replication independent and regulated by Cdc28 and the Mec1 kinase. Embo J 28 1121 1130
57. ZouH
RothsteinR
1997 Holliday junctions accumulate in replication mutants via a RecA homolog-independent mechanism. Cell 90 87 96
58. LopesM
Cotta-RamusinoC
LiberiG
FoianiM
2003 Branch migrating sister chromatid junctions form at replication origins through Rad51/Rad52-independent mechanisms. Mol Cell 12 1499 1510
59. LuCY
TsaiCH
BrillSJ
TengSC
2010 Sumoylation of the BLM ortholog, Sgs1, promotes telomere-telomere recombination in budding yeast. Nucleic Acids Res 38 488 498
60. SanchezY
DesanyBA
JonesWJ
LiuQ
WangB
1996 Regulation of RAD53 by the ATM-like kinases MEC1 and TEL1 in yeast cell cycle checkpoint pathways. Science 271 357 360
61. Cohen-FixO
PetersJM
KirschnerMW
KoshlandD
1996 Anaphase initiation in Saccharomyces cerevisiae is controlled by the APC-dependent degradation of the anaphase inhibitor Pds1p. Genes Dev 10 3081 3093
62. Cohen-FixO
KoshlandD
1997 The anaphase inhibitor of Saccharomyces cerevisiae Pds1p is a target of the DNA damage checkpoint pathway. Proc Natl Acad Sci U S A 94 14361 14366
63. 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
64. PrakashR
SatoryD
DrayE
PapushaA
SchellerJ
2009 Yeast Mph1 helicase dissociates Rad51-made D-loops: implications for crossover control in mitotic recombination. Genes Dev 23 67 79
65. XiongL
ChenXL
SilverHR
AhmedNT
JohnsonES
2009 Deficient SUMO attachment to Flp recombinase leads to homologous recombination-dependent hyperamplification of the yeast 2 microm circle plasmid. Mol Biol Cell 20 1241 1251
66. MotegiA
KuntzK
MajeedA
SmithS
MyungK
2006 Regulation of gross chromosomal rearrangements by ubiquitin and SUMO ligases in Saccharomyces cerevisiae. Mol Cell Biol 26 1424 1433
67. Fishman-LobellJ
RudinN
HaberJE
1992 Two alternative pathways of double-strand break repair that are kinetically separable and independently modulated. Mol Cell Biol 12 1292 1303
68. PaekAL
KaocharS
JonesH
ElezabyA
ShanksL
2009 Fusion of nearby inverted repeats by a replication-based mechanism leads to formation of dicentric and acentric chromosomes that cause genome instability in budding yeast. Genes Dev 23 2861 2875
69. MizunoK
LambertS
BaldacciG
MurrayJM
CarrAM
2009 Nearby inverted repeats fuse to generate acentric and dicentric palindromic chromosomes by a replication template exchange mechanism. Genes Dev 23 2876 2886
70. IrmischA
AmpatzidouE
MizunoK
O'ConnellMJ
MurrayJM
2009 Smc5/6 maintains stalled replication forks in a recombination-competent conformation. Embo J 28 144 155
71. Bermudez-LopezM
CeschiaA
de PiccoliG
ColominaN
PaseroP
2010 The Smc5/6 complex is required for dissolution of DNA-mediated sister chromatid linkages. Nucleic Acids Res
72. SaponaroM
CallahanD
ZhengX
KrejciL
HaberJE
2010 Cdk1 targets Srs2 to complete synthesis-dependent strand annealing and to promote recombinational repair. PLoS Genet 6 e1000858 doi:10.1371/journal.pgen.1000858
73. SacherM
PfanderB
HoegeC
JentschS
2006 Control of Rad52 recombination activity by double-strand break-induced SUMO modification. Nat Cell Biol 8 1284 1290
74. Torres-RosellJ
SunjevaricI
De PiccoliG
SacherM
Eckert-BouletN
2007 The Smc5-Smc6 complex and SUMO modification of Rad52 regulates recombinational repair at the ribosomal gene locus. Nat Cell Biol 9 923 931
75. OhuchiT
SekiM
BranzeiD
MaedaD
UiA
2008 Rad52 sumoylation and its involvement in the efficient induction of homologous recombination. DNA Repair (Amst) 7 879 889
76. BurgessRC
RahmanS
LisbyM
RothsteinR
ZhaoX
2007 The Slx5-Slx8 complex affects sumoylation of DNA repair proteins and negatively regulates recombination. Mol Cell Biol 27 6153 6162
77. BerginkS
JentschS
2009 Principles of ubiquitin and SUMO modifications in DNA repair. Nature 458 461 467
78. 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
79. 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
80. WachA
BrachatA
PohlmannR
PhilippsenP
1994 New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast 10 1793 1808
81. SpencerF
HugeratY
SimchenG
HurkoO
ConnellyC
1994 Yeast kar1 mutants provide an effective method for YAC transfer to new hosts. Genomics 22 118 126
82. Schober-DitmoreW
BachantJ
2000 Yeast DNA flow cytometry.
DiamondR
DeMaggioS
In Living Color: Protocols in Flow Cytometry and Cell Sorting New York Springer Lab Manuals 455 460
83. WarsiTH
NavarroMS
BachantJ
2008 DNA topoisomerase II is a determinant of the tensile properties of yeast centromeric chromatin and the tension checkpoint. Mol Biol Cell 19 4421 4433
84. ChristiansonTW
SikorskiRS
DanteM
SheroJH
HieterP
1992 Multifunctional yeast high-copy-number shuttle vectors. Gene 110 119 122
85. MankouriHW
CraigTJ
MorganA
2002 SGS1 is a multicopy suppressor of srs2: functional overlap between DNA helicases. Nucleic Acids Res 30 1103 1113
86. DesanyBA
AlcasabasAA
BachantJB
ElledgeSJ
1998 Recovery from DNA replicational stress is the essential function of the S-phase checkpoint pathway. Genes Dev 12 2956 2970
Štítky
Genetika Reprodukčná medicínaČlánok vyšiel v časopise
PLOS Genetics
2011 Číslo 3
- Je „freeze-all“ pro všechny? Odborníci na fertilitu diskutovali na virtuálním summitu
- Gynekologové a odborníci na reprodukční medicínu se sejdou na prvním virtuálním summitu
Najčítanejšie v tomto čísle
- Whole-Exome Re-Sequencing in a Family Quartet Identifies Mutations As the Cause of a Novel Skeletal Dysplasia
- Origin-Dependent Inverted-Repeat Amplification: A Replication-Based Model for Generating Palindromic Amplicons
- FUS Transgenic Rats Develop the Phenotypes of Amyotrophic Lateral Sclerosis and Frontotemporal Lobar Degeneration
- Limited dCTP Availability Accounts for Mitochondrial DNA Depletion in Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE)