#PAGE_PARAMS# #ADS_HEAD_SCRIPTS# #MICRODATA#

DNA polymerase theta suppresses mitotic crossing over


Autoři: Juan Carvajal-Garcia aff001;  K. Nicole Crown aff002;  Dale A. Ramsden aff001;  Jeff Sekelsky aff001
Působiště autorů: Curriculum in Genetics and Molecular Biology, University of North Carolina, Chapel Hill, North Carolina, United States of America aff001;  Department of Biology, Case Western Reserve University, Cleveland, Ohio, United States of America aff002;  Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina, United States of America aff003;  Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina, United States of America aff004;  Integrative Program in Biological and Genome Sciences, University of North Carolina, Chapel Hill, North Carolina, United States of America aff005
Vyšlo v časopise: DNA polymerase theta suppresses mitotic crossing over. PLoS Genet 17(3): e1009267. doi:10.1371/journal.pgen.1009267
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.pgen.1009267

Souhrn

Polymerase theta-mediated end joining (TMEJ) is a chromosome break repair pathway that is able to rescue the lethality associated with the loss of proteins involved in early steps in homologous recombination (e.g., BRCA1/2). This is due to the ability of polymerase theta (Pol θ) to use resected, 3’ single stranded DNA tails to repair chromosome breaks. These resected DNA tails are also the starting substrate for homologous recombination. However, it remains unknown if TMEJ can compensate for the loss of proteins involved in more downstream steps during homologous recombination. Here we show that the Holliday junction resolvases SLX4 and GEN1 are required for viability in the absence of Pol θ in Drosophila melanogaster, and lack of all three proteins results in high levels of apoptosis. Flies deficient in Pol θ and SLX4 are extremely sensitive to DNA damaging agents, and mammalian cells require either Pol θ or SLX4 to survive. Our results suggest that TMEJ and Holliday junction formation/resolution share a common DNA substrate, likely a homologous recombination intermediate, that when left unrepaired leads to cell death. One major consequence of Holliday junction resolution by SLX4 and GEN1 is cancer-causing loss of heterozygosity due to mitotic crossing over. We measured mitotic crossovers in flies after a Cas9-induced chromosome break, and observed that this mutagenic form of repair is increased in the absence of Pol θ. This demonstrates that TMEJ can function upstream of the Holiday junction resolvases to protect cells from loss of heterozygosity. Our work argues that Pol θ can thus compensate for the loss of the Holliday junction resolvases by using homologous recombination intermediates, suppressing mitotic crossing over and preserving the genomic stability of cells.

Klíčová slova:

Apoptosis – DNA damage – DNA repair – Drosophila melanogaster – Genetic causes of cancer – Guide RNA – Heterozygosity – Homozygosity


Zdroje

1. Chapman JR, Taylor MRG, Boulton SJ. Playing the End Game: DNA Double-Strand Break Repair Pathway Choice. Molecular Cell. 2012. pp. 497–510. doi: 10.1016/j.molcel.2012.07.029 22920291

2. Scully R, Panday A, Elango R, Willis NA. DNA double-strand break repair-pathway choice in somatic mammalian cells. Nature Reviews Molecular Cell Biology. 2019. pp. 698–714. doi: 10.1038/s41580-019-0152-0 31263220

3. Chan SH, Yu AM, McVey M. Dual roles for DNA polymerase theta in alternative end-joining repair of double-strand breaks in Drosophila. PLoS Genet. 2010;6: 1–16. doi: 10.1371/journal.pgen.1001005 20617203

4. Roerink SF, Schendel R, Tijsterman M. Polymerase theta-mediated end joining of replication-associated DNA breaks in C. elegans. Genome Res. 2014;24: 954–962. doi: 10.1101/gr.170431.113 24614976

5. Yousefzadeh MJ, Wyatt DW, Takata K ichi, Mu Y, Hensley SC, Tomida J, et al. Mechanism of Suppression of Chromosomal Instability by DNA Polymerase POLQ. PLoS Genet. 2014;10. doi: 10.1371/journal.pgen.1004654 25275444

6. Yu AM, McVey M. Synthesis-dependent microhomology-mediated end joining accounts for multiple types of repair junctions. Nucleic Acids Res. 2010;38: 5706–5717. doi: 10.1093/nar/gkq379 20460465

7. Van Kregten M, De Pater S, Romeijn R, Van Schendel R, Hooykaas PJJ, Tijsterman M. T-DNA integration in plants results from polymerase-θ-mediated DNA repair. Nat Plants. 2016;2. doi: 10.1038/nplants.2016.164 27797358

8. Shima N, Munroe RJ, Schimenti JC. The Mouse Genomic Instability Mutation chaos1 Is an Allele of Polq That Exhibits Genetic Interaction with Atm. Mol Cell Biol. 2004;24: 10381–10389. doi: 10.1128/MCB.24.23.10381-10389.2004 15542845

9. Thyme SB, Schier AF. Polq-Mediated End Joining Is Essential for Surviving DNA Double-Strand Breaks during Early Zebrafish Development. Cell Rep. 2016;15: 707–714. doi: 10.1016/j.celrep.2016.03.072 27149851

10. Boyd JB, Sakaguchi K, Harris P V. mus308 Mutants of Drosophila exhibit hypersensitivity to DNA cross-linking agents and are defective in a deoxyribonuclease. Genetics. 1990;125: 813–819. 2397884

11. Muzzini DM, Plevani P, Boulton SJ, Cassata G, Marini F. Caenorhabditis elegans POLQ-1 and HEL-308 function in two distinct DNA interstrand cross-link repair pathways. DNA Repair (Amst). 2008;7: 941–950. doi: 10.1016/j.dnarep.2008.03.021 18472307

12. Wyatt DW, Feng W, Conlin MP, Yousefzadeh MJ, Roberts SA, Mieczkowski P, et al. Essential Roles for Polymerase θ-Mediated End Joining in the Repair of Chromosome Breaks. Mol Cell. 2016;63: 662–673. doi: 10.1016/j.molcel.2016.06.020 27453047

13. Feng W, Simpson DA, Carvajal-Garcia J, Price BA, Kumar RJ, Mose LE, et al. Genetic determinants of cellular addiction to DNA polymerase theta. Nat Commun. 2019;10. doi: 10.1038/s41467-018-07709-6 30602777

14. Ceccaldi R, Liu JC, Amunugama R, Hajdu I, Primack B, Petalcorin MIR, et al. Homologous-recombination-deficient tumours are dependent on Polθ-mediated repair. Nature. 2015;518: 258–262. doi: 10.1038/nature14184 25642963

15. Mateos-Gomez PA, Gong F, Nair N, Miller KM, Lazzerini-Denchi E, Sfeir A. Mammalian polymerase θ promotes alternative NHEJ and suppresses recombination. Nature. 2015;518: 254–257. doi: 10.1038/nature14157 25642960

16. Higgins GS, Boulton SJ. Beyond PARP—POLθ as an anticancer target. Science (80-). 2018;359: 1217–1218. doi: 10.1126/science.aar5149 29590065

17. Stark JM, Pierce AJ, Oh J, Pastink A, Jasin M. Genetic Steps of Mammalian Homologous Repair with Distinct Mutagenic Consequences. Mol Cell Biol. 2004;24: 9305–9316. doi: 10.1128/MCB.24.21.9305-9316.2004 15485900

18. Zhu X, Dunn JM, Goddard AD, Squire JA, Becker A, Phillips RA, et al. Mechanisms of loss of heterozygosity in retinoblastoma. Cytogenet Genome Res. 1992;59: 248–252. doi: 10.1159/000133261 1544317

19. Luo G, Santoro IM, McDaniel LD, Nishijima I, Mills M, Youssoufian H, et al. Cancer predisposition caused by elevated mitotic recombination in Bloom mice. Nat Genet. 2000;26: 424–429. doi: 10.1038/82548 11101838

20. Lemée F, Bergoglio V, Fernandez-Vidal A, Machado-Silva A, Pillaire M-J, Bieth A, et al. DNA polymerase theta up-regulation is associated with poor survival in breast cancer, perturbs DNA replication, and promotes genetic instability. Proc Natl Acad Sci U S A. 2010;107: 13390–5. doi: 10.1073/pnas.0910759107 20624954

21. Carvajal-Garcia J, Cho JE, Carvajal-Garcia P, Feng W, Wood RD, Sekelsky J, et al. Mechanistic basis for microhomology identification and genome scarring by polymerase theta. Proc Natl Acad Sci U S A. 2020;117: 8476–8485. doi: 10.1073/pnas.1921791117 32234782

22. Kamp JA, van Schendel R, Dilweg IW, Tijsterman M. BRCA1-associated structural variations are a consequence of polymerase theta-mediated end-joining. Nat Commun. 2020;11. doi: 10.1038/s41467-020-17455-3 32680986

23. Fricke WM, Brill SJ. Slx1—Slx4 is a second structure-specific endonuclease functionally redundant with Sgs1—Top3. Genes Dev. 2003;17: 1768–1778. doi: 10.1101/gad.1105203 12832395

24. Andersen SL, Bergstralh DT, Kohl KP, LaRocque JR, Moore CB, Sekelsky J. Drosophila MUS312 and the Vertebrate Ortholog BTBD12 Interact with DNA Structure-Specific Endonucleases in DNA Repair and Recombination. Mol Cell. 2009;35: 128–135. doi: 10.1016/j.molcel.2009.06.019 19595722

25. Muñoz IM, Hain K, Déclais AC, Gardiner M, Toh GW, Sanchez-Pulido L, et al. Coordination of Structure-Specific Nucleases by Human SLX4/BTBD12 Is Required for DNA Repair. Mol Cell. 2009;35: 116–127. doi: 10.1016/j.molcel.2009.06.020 19595721

26. Svendsen JM, Smogorzewska A, Sowa ME, O’Connell BC, Gygi SP, Elledge SJ, et al. Mammalian BTBD12/SLX4 Assembles A Holliday Junction Resolvase and Is Required for DNA Repair. Cell. 2009;138: 63–77. doi: 10.1016/j.cell.2009.06.030 19596235

27. Fekairi S, Scaglione S, Chahwan C, Taylor ER, Tissier A, Coulon S, et al. Human SLX4 Is a Holliday Junction Resolvase Subunit that Binds Multiple DNA Repair/Recombination Endonucleases. Cell. 2009;138: 78–89. doi: 10.1016/j.cell.2009.06.029 19596236

28. Ip SCY, Rass U, Blanco MG, Flynn HR, Skehel JM, West SC. Identification of Holliday junction resolvases from humans and yeast. Nature. 2008;456: 357–361. doi: 10.1038/nature07470 19020614

29. Wyatt HDM, Laister RC, Martin SR, Arrowsmith CH, West SC. The SMX DNA Repair Tri-nuclease. Mol Cell. 2017;65: 848–860.e11. doi: 10.1016/j.molcel.2017.01.031 28257701

30. Chan YW, West S. GEN1 promotes Holliday junction resolution by a coordinated nick and counter-nick mechanism. Nucleic Acids Res. 2015;43: 10882–10892. doi: 10.1093/nar/gkv1207 26578604

31. Bellendir SP, Rognstad DJ, Morris LP, Zapotoczny G, Walton WG, Redinbo MR, et al. Substrate preference of Gen endonucleases highlights the importance of branched structures as DNA damage repair intermediates. Nucleic Acids Res. 2017;45: 5333–5348. doi: 10.1093/nar/gkx214 28369583

32. Brinkman EK, Chen T, Amendola M, Van Steensel B. Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res. 2014;42. doi: 10.1093/nar/gku936 25300484

33. Song Z, McCall K, Steller H. DCP-1, a Drosophila cell death protease essential for development. Science (80-). 1997;275: 536–540. doi: 10.1126/science.275.5299.536 8999799

34. Adams MD, McVey M, Sekelsky JJ. Drosophila BLM in double-strand break repair by synthesis-dependent strand annealing. Science (80-). 2003;299: 265–267. doi: 10.1126/science.1077198 12522255

35. McVey M, Andersen SL, Broze Y, Sekelsky J. Multiple functions of drosophila BLM helicase in maintenance of genome stability. Genetics. 2007;176: 1979–1992. doi: 10.1534/genetics.106.070052 17507683

36. Kenny Kuo H, McMahan S, Rota CM, Kohl KP, Sekelsky J. Drosophila FANCM helicase prevents spontaneous mitotic crossovers generated by the MUS81 and SLX1 nucleases. Genetics. 2014;198: 935–945. doi: 10.1534/genetics.114.168096 25205745

37. Schrempf A, Slyskova J, Loizou JI. Targeting the DNA Repair Enzyme Polymerase θ in Cancer Therapy. Trends in Cancer. 2020;xx: 1–14. doi: 10.1016/j.trecan.2020.09.007 33109489

38. Wei DS, Rong YS. A genetic screen for DNA double-strand break repair mutations in Drosophila. Genetics. 2007;177: 63–77. doi: 10.1534/genetics.107.077693 17660539

39. Kelso AA, Lopezcolorado FW, Bhargava R, Stark JM. Distinct roles of RAD52 and POLQ in chromosomal break repair and replication stress response. PLoS Genet. 2019;15. doi: 10.1371/journal.pgen.1008319 31381562

40. Davis L, Khoo KJ, Zhang Y, Maizels N. POLQ suppresses interhomolog recombination and loss of heterozygosity at targeted DNA breaks. Proc Natl Acad Sci U S A. 2020;117: 22900–22909. doi: 10.1073/pnas.2008073117 32873648

41. Klovstad M, Abdu U, Schüpbach T. Drosophila brca2 is required for mitotic and meiotic DNA repair and efficient activation of the meiotic recombination checkpoint. PLoS Genet. 2008;4. doi: 10.1371/journal.pgen.0040031 18266476

42. Thomas AM, Hui C, South A, McVey M. Common variants of drosophila melanogaster Cyp6d2 cause camptothecin sensitivity and synergize with loss of Brca2. G3 Genes, Genomes, Genet. 2013;3: 91–99. doi: 10.1534/g3.112.003996 23316441

43. Beagan K, Armstrong RL, Witsell A, Roy U, Renedo N, Baker AE, et al. Drosophila DNA polymerase theta utilizes both helicase-like and polymerase domains during microhomology-mediated end joining and interstrand crosslink repair. PLoS Genet. 2017;13. doi: 10.1371/journal.pgen.1006813 28542210

44. Yildiz Ö, Majumder S, Kramer B, Sekelsky JJ. Drosophila MUS312 interacts with the nucleotide excision repair endonuclease MEI-9 to generate meiotic crossovers. Mol Cell. 2002;10: 1503–1509. doi: 10.1016/s1097-2765(02)00782-7 12504024

45. Andersen SL, Kuo HK, Savukoski D, Brodsky MH, Sekelsky J. Three structure-selective endonucleases are essential in the absence of BLM helicase in Drosophila. PLoS Genet. 2011;7. doi: 10.1371/journal.pgen.1002315 22022278

46. Trowbridge K, McKim K, Brill SJ, Sekelsky J. Synthetic lethality of drosophila in the absence of the MUS81 endonuclease and the DmBlm helicase is associated with elevated apoptosis. Genetics. 2007;176: 1993–2001. doi: 10.1534/genetics.106.070060 17603121

47. Port F, Chen H-M, Lee T, Bullock SL. Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila. Proc Natl Acad Sci. 2014;111: E2967–E2976. doi: 10.1073/pnas.1405500111 25002478

48. Gratz SJ, Cummings AM, Nguyen JN, Hamm DC, Donohue LK, Harrison MM, et al. Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease. Genetics. 2013. pp. 1029–1035. doi: 10.1534/genetics.113.152710 23709638

49. Sekelsky J. DNA repair in Drosophila: Mutagens, models, and missing genes. Genetics. 2017;205: 471–490. doi: 10.1534/genetics.116.186759 28154196

50. Janetzko K, Rink G, Hecker A, Bieback K, Klüter H, Bugert P. A single-tube real-time PCR assay for mycoplasma detection as a routine quality control of cell therapeutics. Transfus Med Hemotherapy. 2014;41: 83–89. doi: 10.1159/000357096 24659951

51. Battaglia M, Pozzi D, Crimaldi S, Parasassi T. Hoechst 33258 staining for detecting mycoplasma contamination in cell cultures: A method for reducing fluorescence photobleaching. Biotech Histochem. 1994;69: 152–156. doi: 10.3109/10520299409106277 7520758

52. Lin S, Staahl BT, Alla RK, Doudna JA. Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. Elife. 2014;3: e04766. doi: 10.7554/eLife.04766 25497837

53. Franken NAP, Rodermond HM, Stap J, Haveman J, van Bree C. Clonogenic assay of cells in vitro. Nat Protoc. 2006;1: 2315–2319. doi: 10.1038/nprot.2006.339 17406473

54. Dewey EB, Johnston CA. Diverse mitotic functions of the cytoskeletal cross-linking protein Shortstop suggest a role in Dynein/Dynactin activity. Mol Biol Cell. 2017;28: 2555–2565. doi: 10.1091/mbc.E17-04-0219 28747439


Článek vyšel v časopise

PLOS Genetics


2021 Číslo 3
Nejčtenější tento týden
Nejčtenější v tomto čísle
Kurzy Podcasty Doporučená témata Časopisy
Přihlášení
Zapomenuté heslo

Zadejte e-mailovou adresu, se kterou jste vytvářel(a) účet, budou Vám na ni zaslány informace k nastavení nového hesla.

Přihlášení

Nemáte účet?  Registrujte se

#ADS_BOTTOM_SCRIPTS#