Heteroduplex DNA Position Defines the Roles of the Sgs1, Srs2, and Mph1 Helicases in Promoting Distinct Recombination Outcomes

English version České info

The contributions of the Sgs1, Mph1, and Srs2 DNA helicases during mitotic double-strand break (DSB) repair in yeast were investigated using a gap-repair assay. A diverged chromosomal substrate was used as a repair template for the gapped plasmid, allowing mismatch-containing heteroduplex DNA (hDNA) formed during recombination to be monitored. Overall DSB repair efficiencies and the proportions of crossovers (COs) versus noncrossovers (NCOs) were determined in wild-type and helicase-defective strains, allowing the efficiency of CO and NCO production in each background to be calculated. In addition, the products of individual NCO events were sequenced to determine the location of hDNA. Because hDNA position is expected to differ depending on whether a NCO is produced by synthesis-dependent-strand-annealing (SDSA) or through a Holliday junction (HJ)–containing intermediate, its position allows the underlying molecular mechanism to be inferred. Results demonstrate that each helicase reduces the proportion of CO recombinants, but that each does so in a fundamentally different way. Mph1 does not affect the overall efficiency of gap repair, and its loss alters the CO-NCO by promoting SDSA at the expense of HJ–containing intermediates. By contrast, Sgs1 and Srs2 are each required for efficient gap repair, strongly promoting NCO formation and having little effect on CO efficiency. hDNA analyses suggest that all three helicases promote SDSA, and that Sgs1 and Srs2 additionally dismantle HJ–containing intermediates. The hDNA data are consistent with the proposed role of Sgs1 in the dissolution of double HJs, and we propose that Srs2 dismantles nicked HJs.

Vyšlo v časopise: Heteroduplex DNA Position Defines the Roles of the Sgs1, Srs2, and Mph1 Helicases in Promoting Distinct Recombination Outcomes. PLoS Genet 9(3): e32767. doi:10.1371/journal.pgen.1003340
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1003340

Souhrn

Zdroje

1. PaquesF, HaberJE (1999) Multiple pathways of recombination induced by double-strand breaks in Saccharomyces cerevisiae. Microbiology and molecular biology reviews : MMBR 63 : 349–404.

2. San FilippoJ, SungP, KleinH (2008) Mechanism of eukaryotic homologous recombination. Annual review of biochemistry 77 : 229–257.

3. SymingtonLS (2002) Role of RAD52 epistasis group genes in homologous recombination and double-strand break repair. Microbiology and molecular biology reviews : MMBR 66 : 630–670, table of contents.

4. SymingtonLS, GautierJ (2011) Double-strand break end resection and repair pathway choice. Annual review of genetics 45 : 247–271.

5. FergusonDO, HollomanWK (1996) Recombinational repair of gaps in DNA is asymmetric in Ustilago maydis and can be explained by a migrating D-loop model. Proceedings of the National Academy of Sciences of the United States of America 93 : 5419–5424.

6. SzostakJW, Orr-WeaverTL, RothsteinRJ, StahlFW (1983) The double-strand-break repair model for recombination. Cell 33 : 25–35.

7. SymingtonLS, KangLE, MoreauS (2000) Alteration of gene conversion tract length and associated crossing over during plasmid gap repair in nuclease-deficient strains of Saccharomyces cerevisiae. Nucleic Acids Research 28 : 4649–4656.

8. Welz-VoegeleC, Jinks-RobertsonS (2008) Sequence divergence impedes crossover more than noncrossover events during mitotic gap repair in yeast. Genetics 179 : 1251–1262.

9. MankouriHW, HicksonID (2007) The RecQ helicase-topoisomerase III-Rmi1 complex: a DNA structure-specific ‘dissolvasome’? Trends in biochemical sciences 32 : 538–546.

10. NassifN, PenneyJ, PalS, EngelsWR, GloorGB (1994) Efficient copying of nonhomologous sequences from ectopic sites via P-element-induced gap repair. Molecular and cellular biology 14 : 1613–1625.

11. HeyerWD, EhmsenKT, LiuJ (2010) Regulation of homologous recombination in eukaryotes. Annual review of genetics 44 : 113–139.

12. IraG, MalkovaA, LiberiG, FoianiM, HaberJE (2003) Srs2 and Sgs1-Top3 suppress crossovers during double-strand break repair in yeast. Cell 115 : 401–411.

13. PrakashR, SatoryD, DrayE, PapushaA, SchellerJ, et al. (2009) Yeast Mph1 helicase dissociates Rad51-made D-loops: implications for crossover control in mitotic recombination. Genes Dev 23 : 67–79.

14. LawrenceCW, ChristensenRB (1979) Metabolic suppressors of trimethoprim and ultraviolet light sensitivities of Saccharomyces cerevisiae rad6 mutants. Journal of bacteriology 139 : 866–876.

15. SchiestlRH, PrakashS, PrakashL (1990) The SRS2 suppressor of rad6 mutations of Saccharomyces cerevisiae acts by channeling DNA lesions into the RAD52 DNA repair pathway. Genetics 124 : 817–831.

16. RongL, KleinHL (1993) Purification and characterization of the SRS2 DNA helicase of the yeast Saccharomyces cerevisiae. The Journal of biological chemistry 268 : 1252–1259.

17. AguileraA, KleinHL (1988) Genetic control of intrachromosomal recombination in Saccharomyces cerevisiae. I. Isolation and genetic characterization of hyper-recombination mutations. Genetics 119 : 779–790.

18. KrejciL, Van KomenS, LiY, VillemainJ, ReddyMS, et al. (2003) DNA helicase Srs2 disrupts the Rad51 presynaptic filament. Nature 423 : 305–309.

19. VeauteX, JeussetJ, SoustelleC, KowalczykowskiSC, Le CamE, et al. (2003) The Srs2 helicase prevents recombination by disrupting Rad51 nucleoprotein filaments. Nature 423 : 309–312.

20. AylonY, LiefshitzB, Bitan-BaninG, KupiecM (2003) Molecular dissection of mitotic recombination in the yeast Saccharomyces cerevisiae. Molecular and cellular biology 23 : 1403–1417.

21. DupaigneP, Le BretonC, FabreF, GangloffS, Le CamE, et al. (2008) The Srs2 helicase activity is stimulated by Rad51 filaments on dsDNA: implications for crossover incidence during mitotic recombination. Mol Cell 29 : 243–254.

22. WuY, KantakeN, SugiyamaT, KowalczykowskiSC (2008) Rad51 protein controls Rad52-mediated DNA annealing. The Journal of biological chemistry 283 : 14883–14892.

23. GangloffS, McDonaldJP, BendixenC, ArthurL, RothsteinR (1994) The yeast type I topoisomerase Top3 interacts with Sgs1, a DNA helicase homolog: a potential eukaryotic reverse gyrase. Molecular and cellular biology 14 : 8391–8398.

24. CejkaP, KowalczykowskiSC (2010) The full-length Saccharomyces cerevisiae Sgs1 protein is a vigorous DNA helicase that preferentially unwinds holliday junctions. The Journal of biological chemistry 285 : 8290–8301.

25. BernsteinKA, GangloffS, RothsteinR (2010) The RecQ DNA helicases in DNA repair. Annual review of genetics 44 : 393–417.

26. MimitouEP, SymingtonLS (2009) DNA end resection: many nucleases make light work. DNA Repair (Amst) 8 : 983–995.

27. CejkaP, PlankJL, BachratiCZ, HicksonID, KowalczykowskiSC (2010) Rmi1 stimulates decatenation of double Holliday junctions during dissolution by Sgs1-Top3. Nature structural & molecular biology 17 : 1377–1382.

28. MankouriHW, AshtonTM, HicksonID (2011) Holliday junction-containing DNA structures persist in cells lacking Sgs1 or Top3 following exposure to DNA damage. Proceedings of the National Academy of Sciences of the United States of America 108 : 4944–4949.

29. EntianKD, SchusterT, HegemannJH, BecherD, FeldmannH, et al. (1999) Functional analysis of 150 deletion mutants in Saccharomyces cerevisiae by a systematic approach. Molecular & general genetics : MGG 262 : 683–702.

30. MeeteiAR, MedhurstAL, LingC, XueY, SinghTR, et al. (2005) A human ortholog of archaeal DNA repair protein Hef is defective in Fanconi anemia complementation group M. Nature genetics 37 : 958–963.

31. MosedaleG, NiedzwiedzW, AlpiA, PerrinaF, Pereira-LealJB, et al. (2005) The vertebrate Hef ortholog is a component of the Fanconi anemia tumor-suppressor pathway. Nature structural & molecular biology 12 : 763–771.

32. SchurerKA, RudolphC, UlrichHD, KramerW (2004) Yeast MPH1 gene functions in an error-free DNA damage bypass pathway that requires genes from homologous recombination, but not from postreplicative repair. Genetics 166 : 1673–1686.

33. ZhengXF, PrakashR, SaroD, LongerichS, NiuH, et al. (2011) Processing of DNA structures via DNA unwinding and branch migration by the S. cerevisiae Mph1 protein. DNA Repair 10 : 1034–1043.

34. TayYD, SidebothamJM, WuL (2010) Mph1 requires mismatch repair-independent and -dependent functions of MutSalpha to regulate crossover formation during homologous recombination repair. Nucleic Acids Research 38 : 1889–1901.

35. MitchelK, ZhangH, Welz-VoegeleC, Jinks-RobertsonS (2010) Molecular structures of crossover and noncrossover intermediates during gap repair in yeast: implications for recombination. Mol Cell 38 : 211–222.

36. ColavitoS, Macris-KissM, SeongC, GleesonO, GreeneEC, et al. (2009) Functional significance of the Rad51-Srs2 complex in Rad51 presynaptic filament disruption. Nucleic acids research 37 : 6754–6764.

37. SunW, NandiS, OsmanF, AhnJS, JakovleskaJ, et al. (2008) The FANCM ortholog Fml1 promotes recombination at stalled replication forks and limits crossing over during DNA double-strand break repair. Molecular cell 32 : 118–128.

38. SebestaM, BurkovicsP, HaracskaL, KrejciL (2011) Reconstitution of DNA repair synthesis in vitro and the role of polymerase and helicase activities. DNA Repair 10 : 567–576.

39. GangloffS, SoustelleC, FabreF (2000) Homologous recombination is responsible for cell death in the absence of the Sgs1 and Srs2 helicases. Nature genetics 25 : 192–194.

40. LaRocqueJR, StarkJM, OhJ, BojilovaE, YusaK, et al. (2011) Interhomolog recombination and loss of heterozygosity in wild-type and Bloom syndrome helicase (BLM)-deficient mammalian cells. Proceedings of the National Academy of Sciences of the United States of America 108 : 11971–11976.

41. McVeyM, LarocqueJR, AdamsMD, SekelskyJJ (2004) Formation of deletions during double-strand break repair in Drosophila DmBlm mutants occurs after strand invasion. Proceedings of the National Academy of Sciences of the United States of America 101 : 15694–15699.

42. WuL, HicksonID (2003) The Bloom's syndrome helicase suppresses crossing over during homologous recombination. Nature 426 : 870–874.

43. PlankJL, WuJ, HsiehTS (2006) Topoisomerase IIIalpha and Bloom's helicase can resolve a mobile double Holliday junction substrate through convergent branch migration. Proceedings of the National Academy of Sciences of the United States of America 103 : 11118–11123.

44. DayaniY, SimchenG, LichtenM (2011) Meiotic recombination intermediates are resolved with minimal crossover formation during return-to-growth, an analogue of the mitotic cell cycle. PLoS Genet 7: e1002083 doi:10.1371/journal.pgen.1002083.

45. LiberiG, MaffiolettiG, LuccaC, ChioloI, BaryshnikovaA, et al. (2005) Rad51-dependent DNA structures accumulate at damaged replication forks in sgs1 mutants defective in the yeast ortholog of BLM RecQ helicase. Genes & development 19 : 339–350.

46. AdamsMD, McVeyM, SekelskyJJ (2003) Drosophila BLM in double-strand break repair by synthesis-dependent strand annealing. Science 299 : 265–267.

47. McVeyM, AndersenSL, BrozeY, SekelskyJ (2007) Multiple functions of Drosophila BLM helicase in maintenance of genome stability. Genetics 176 : 1979–1992.

48. van BrabantAJ, YeT, SanzM, GermanIJ, EllisNA, et al. (2000) Binding and melting of D-loops by the Bloom syndrome helicase. Biochemistry 39 : 14617–14625.

49. BachratiCZ, BortsRH, HicksonID (2006) Mobile D-loops are a preferred substrate for the Bloom's syndrome helicase. Nucleic acids research 34 : 2269–2279.

50. ChenCF, BrillSJ (2010) An essential DNA strand-exchange activity is conserved in the divergent N-termini of BLM orthologs. The EMBO journal 29 : 1713–1725.

51. De MuytA, JessopL, KolarE, SourirajanA, ChenJ, et al. (2012) BLM helicase ortholog Sgs1 is a central regulator of meiotic recombination intermediate metabolism. Molecular cell 46 : 43–53.

52. ZakharyevichK, TangS, MaY, HunterN (2012) Delineation of joint molecule resolution pathways in meiosis identifies a crossover-specific resolvase. Cell 149 : 334–347.

53. RobertT, DervinsD, FabreF, GangloffS (2006) Mrc1 and Srs2 are major actors in the regulation of spontaneous crossover. The EMBO journal 25 : 2837–2846.

54. BlanckS, KobbeD, HartungF, FenglerK, FockeM, et al. (2009) A SRS2 homolog from Arabidopsis thaliana disrupts recombinogenic DNA intermediates and facilitates single strand annealing. Nucleic acids research 37 : 7163–7176.

55. PaquesF, LeungWY, HaberJE (1998) Expansions and contractions in a tandem repeat induced by double-strand break repair. Molecular and cellular biology 18 : 2045–2054.

56. MiuraT, YamanaY, UsuiT, OgawaHI, YamamotoMT, et al. (2012) Homologous recombination via synthesis-dependent strand annealing in yeast requires the Irc20 and Srs2 DNA helicases. Genetics 191 : 65–78.

57. InbarO, LiefshitzB, BitanG, KupiecM (2000) The relationship between homology length and crossing over during the repair of a broken chromosome. The Journal of biological chemistry 275 : 30833–30838.

58. MazonG, LamAF, HoCK, KupiecM, SymingtonLS (2012) The Rad1-Rad10 nuclease promotes chromosome translocations between dispersed repeats. Nature structural & molecular biology 19 : 964–971.

59. SikorskiRS, HieterP (1989) A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122 : 19–27.