Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

RPA antagonizes microhomology-mediated repair of DNA double-strand breaks

Nature Structural & Molecular Biology volume 21, pages 405–412 (2014)Cite this article

Subjects

Abstract

Microhomology-mediated end joining (MMEJ) is a Ku- and ligase IV–independent mechanism for the repair of DNA double-strand breaks that contributes to chromosome rearrangements. Here we used a chromosomal end-joining assay to determine the genetic requirements for MMEJ in Saccharomyces cerevisiae. We found that end resection influences the ability to expose microhomologies; however, it is not rate limiting for MMEJ in wild-type cells. The frequency of MMEJ increased by up to 350-fold in rfa1 hypomorphic mutants, suggesting that replication protein A (RPA) bound to the single-stranded DNA (ssDNA) overhangs formed by resection prevents spontaneous annealing between microhomologies. In vitro, the mutant RPA complexes were unable to fully extend ssDNA and were compromised in their ability to prevent spontaneous annealing. We propose that the helix-destabilizing activity of RPA channels ssDNA intermediates from mutagenic MMEJ to error-free homologous recombination, thus preserving genome integrity.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Role of resection initiation and extensive resection in end-joining repair.
Figure 2: Annealing between MHs is prevented by RPA.
Figure 3: RPA mutants are defective for ssDNA binding and disruption of secondary structure.
Figure 4: HR and MMEJ are competing mechanisms.
Figure 5: Regulation of repair pathway choice by Sae2 and RPA.

Similar content being viewed by others

References

  1. Lieber, M.R. NHEJ and its backup pathways in chromosomal translocations. Nat. Struct. Mol. Biol. 17, 393–395 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. McVey, M. & Lee, S.E. MMEJ repair of double-strand breaks (director's cut): deleted sequences and alternative endings. Trends Genet. 24, 529–538 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Boboila, C., Alt, F.W. & Schwer, B. Classical and alternative end-joining pathways for repair of lymphocyte-specific and general DNA double-strand breaks. Adv. Immunol. 116, 1–49 (2012).

    CAS  PubMed  Google Scholar 

  4. Ma, J.L., Kim, E.M., Haber, J.E. & Lee, S.E. Yeast Mre11 and Rad1 proteins define a Ku-independent mechanism to repair double-strand breaks lacking overlapping end sequences. Mol. Cell. Biol. 23, 8820–8828 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Symington, L.S. Role of RAD52 epistasis group genes in homologous recombination and double-strand break repair. Microbiol. Mol. Biol. Rev. 66, 630–670 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Symington, L.S. & Gautier, J. Double-strand break end resection and repair pathway choice. Annu. Rev. Genet. 45, 247–271 (2011).

    CAS  PubMed  Google Scholar 

  7. Mimitou, E.P. & Symington, L.S. Sae2, Exo1 and Sgs1 collaborate in DNA double-strand break processing. Nature 455, 770–774 (2008).

    CAS  PubMed  Google Scholar 

  8. Shim, E.Y. et al. Saccharomyces cerevisiae Mre11/Rad50/Xrs2 and Ku proteins regulate association of Exo1 and Dna2 with DNA breaks. EMBO J. 29, 3370–3380 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Zhu, Z., Chung, W.H., Shim, E.Y., Lee, S.E. & Ira, G. Sgs1 helicase and two nucleases Dna2 and Exo1 resect DNA double-strand break ends. Cell 134, 981–994 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Mimitou, E.P. & Symington, L.S. DNA end resection: many nucleases make light work. DNA Repair (Amst.) 8, 983–995 (2009).

    CAS  Google Scholar 

  11. Buis, J. et al. Mre11 nuclease activity has essential roles in DNA repair and genomic stability distinct from ATM activation. Cell 135, 85–96 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Langerak, P., Mejia-Ramirez, E., Limbo, O. & Russell, P. Release of Ku and MRN from DNA ends by Mre11 nuclease activity and Ctp1 is required for homologous recombination repair of double-strand breaks. PLoS Genet. 7, e1002271 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Sartori, A.A. et al. Human CtIP promotes DNA end resection. Nature 450, 509–514 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Bennardo, N., Cheng, A., Huang, N. & Stark, J.M. Alternative-NHEJ is a mechanistically distinct pathway of mammalian chromosome break repair. PLoS Genet. 4, e1000110 (2008).

    PubMed  PubMed Central  Google Scholar 

  15. Lee-Theilen, M., Matthews, A.J., Kelly, D., Zheng, S. & Chaudhuri, J. CtIP promotes microhomology-mediated alternative end joining during class-switch recombination. Nat. Struct. Mol. Biol. 18, 75–79 (2011).

    CAS  PubMed  Google Scholar 

  16. Truong, L.N. et al. Microhomology-mediated end joining and homologous recombination share the initial end resection step to repair DNA double-strand breaks in mammalian cells. Proc. Natl. Acad. Sci. USA 110, 7720–7725 (2013).

    CAS  PubMed  Google Scholar 

  17. Zhang, Y. & Jasin, M. An essejntial role for CtIP in chromosomal translocation formation through an alternative end-joining pathway. Nat. Struct. Mol. Biol. 18, 80–84 (2011).

    CAS  PubMed  Google Scholar 

  18. Dinkelmann, M. et al. Multiple functions of MRN in end-joining pathways during isotype class switching. Nat. Struct. Mol. Biol. 16, 808–813 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Lee, K. & Lee, S.E. Saccharomyces cerevisiae Sae2- and Tel1-dependent single-strand DNA formation at DNA break promotes microhomology-mediated end joining. Genetics 176, 2003–2014 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Xie, A., Kwok, A. & Scully, R. Role of mammalian Mre11 in classical and alternative nonhomologous end joining. Nat. Struct. Mol. Biol. 16, 814–818 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Rass, E. et al. Role of Mre11 in chromosomal nonhomologous end joining in mammalian cells. Nat. Struct. Mol. Biol. 16, 819–824 (2009).

    CAS  PubMed  Google Scholar 

  22. Mortensen, U.H., Bendixen, C., Sunjevaric, I. & Rothstein, R. DNA strand annealing is promoted by the yeast Rad52 protein. Proc. Natl. Acad. Sci. USA 93, 10729–10734 (1996).

    CAS  PubMed  Google Scholar 

  23. Sugawara, N. & Haber, J.E. Characterization of double-strand break–induced recombination: homology requirements and single-stranded DNA formation. Mol. Cell. Biol. 12, 563–575 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Villarreal, D.D. et al. Microhomology directs diverse DNA break repair pathways and chromosomal translocations. PLoS Genet. 8, e1003026 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Daley, J.M. & Wilson, T.E. Rejoining of DNA double-strand breaks as a function of overhang length. Mol. Cell. Biol. 25, 896–906 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Wold, M.S. Replication protein A: a heterotrimeric, single-stranded DNA-binding protein required for eukaryotic DNA metabolism. Annu. Rev. Biochem. 66, 61–92 (1997).

    CAS  PubMed  Google Scholar 

  27. Sugiyama, T., New, J.H. & Kowalczykowski, S.C. DNA annealing by RAD52 protein is stimulated by specific interaction with the complex of replication protein A and single-stranded DNA. Proc. Natl. Acad. Sci. USA 95, 6049–6054 (1998).

    CAS  PubMed  Google Scholar 

  28. Smith, J. & Rothstein, R. A mutation in the gene encoding the Saccharomyces cerevisiae single-stranded DNA–binding protein Rfa1 stimulates a RAD52-independent pathway for direct-repeat recombination. Mol. Cell. Biol. 15, 1632–1641 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Smith, J. & Rothstein, R. An allele of RFA1 suppresses RAD52-dependent double-strand break repair in Saccharomyces cerevisiae. Genetics 151, 447–458 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Soustelle, C., Vedel, M., Kolodner, R. & Nicolas, A. Replication protein A is required for meiotic recombination in Saccharomyces cerevisiae. Genetics 161, 535–547 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Umezu, K., Sugawara, N., Chen, C., Haber, J.E. & Kolodner, R.D. Genetic analysis of yeast RPA1 reveals its multiple functions in DNA metabolism. Genetics 148, 989–1005 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Chen, H., Lisby, M. & Symington, L. RPA coordinates DNA end resection and prevents formation of DNA hairpins. Mol. Cell 50, 589–600 (2013).

    CAS  PubMed  Google Scholar 

  33. Milne, G.T., Jin, S., Shannon, K.B. & Weaver, D.T. Mutations in two Ku homologs define a DNA end-joining repair pathway in Saccharomyces cerevisiae. Mol. Cell. Biol. 16, 4189–4198 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Moore, J.K. & Haber, J.E. Cell cycle and genetic requirements of two pathways of nonhomologous end-joining repair of double-strand breaks in Saccharomyces cerevisiae. Mol. Cell. Biol. 16, 2164–2173 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Kantake, N., Sugiyama, T., Kolodner, R.D. & Kowalczykowski, S.C. The recombination-deficient mutant RPA (rfa1-t11) is displaced slowly from single-stranded DNA by Rad51 protein. J. Biol. Chem. 278, 23410–23417 (2003).

    CAS  PubMed  Google Scholar 

  36. Lee, S.E. et al. Saccharomyces Ku70, mre11/rad50 and RPA proteins regulate adaptation to G2/M arrest after DNA damage. Cell 94, 399–409 (1998).

    CAS  PubMed  Google Scholar 

  37. Lobachev, K.S., Gordenin, D.A. & Resnick, M.A. The Mre11 complex is required for repair of hairpin-capped double-strand breaks and prevention of chromosome rearrangements. Cell 108, 183–193 (2002).

    CAS  PubMed  Google Scholar 

  38. Rattray, A.J., Shafer, B.K., Neelam, B. & Strathern, J.N. A mechanism of palindromic gene amplification in Saccharomyces cerevisiae. Genes Dev. 19, 1390–1399 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Gibb, B., Silverstein, T.D., Finkelstein, I.J. & Greene, E.C. Single-stranded DNA curtains for real-time single-molecule visualization of protein-nucleic acid interactions. Anal. Chem. 84, 7607–7612 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Gibb, B. et al. Concentration-dependent exchange of replication protein a on single-stranded DNA revealed by single-molecule imaging. PLoS ONE 9, e87922 (2014).

    PubMed  PubMed Central  Google Scholar 

  41. Graham, J.S., Johnson, R.C. & Marko, J.F. Concentration-dependent exchange accelerates turnover of proteins bound to double-stranded DNA. Nucleic Acids Res. 39, 2249–2259 (2011).

    CAS  PubMed  Google Scholar 

  42. Liu, P., Carvalho, C.M., Hastings, P.J. & Lupski, J.R. Mechanisms for recurrent and complex human genomic rearrangements. Curr. Opin. Genet. Dev. 22, 211–220 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Stephens, P.J. et al. Complex landscapes of somatic rearrangement in human breast cancer genomes. Nature 462, 1005–1010 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Stephens, P.J. et al. Massive genomic rearrangement acquired in a single catastrophic event during cancer development. Cell 144, 27–40 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Pâques, F. & Haber, J.E. Multiple pathways of recombination induced by double-strand breaks in Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 63, 349–404 (1999).

    PubMed  PubMed Central  Google Scholar 

  46. Audebert, M., Salles, B. & Calsou, P. Involvement of poly(ADP-ribose) polymerase-1 and XRCC1/DNA ligase III in an alternative route for DNA double-strand breaks rejoining. J. Biol. Chem. 279, 55117–55126 (2004).

    CAS  PubMed  Google Scholar 

  47. Mansour, W.Y., Rhein, T. & Dahm-Daphi, J. The alternative end-joining pathway for repair of DNA double-strand breaks requires PARP1 but is not dependent upon microhomologies. Nucleic Acids Res. 38, 6065–6077 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Wang, M. et al. PARP-1 and Ku compete for repair of DNA double strand breaks by distinct NHEJ pathways. Nucleic Acids Res. 34, 6170–6182 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Liang, L. et al. Human DNA ligases I and III, but not ligase IV, are required for microhomology-mediated end joining of DNA double-strand breaks. Nucleic Acids Res. 36, 3297–3310 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Simsek, D. et al. DNA ligase III promotes alternative nonhomologous end-joining during chromosomal translocation formation. PLoS Genet. 7, e1002080 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Wang, H. et al. DNA ligase III as a candidate component of backup pathways of nonhomologous end joining. Cancer Res. 65, 4020–4030 (2005).

    CAS  PubMed  Google Scholar 

  52. Matsuzaki, K., Terasawa, M., Iwasaki, D., Higashide, M. & Shinohara, M. Cyclin-dependent kinase-dependent phosphorylation of Lif1 and Sae2 controls imprecise nonhomologous end joining accompanied by double-strand break resection. Genes Cells 17, 473–493 (2012).

    CAS  PubMed  Google Scholar 

  53. Shibata, A. et al. Factors determining DNA double-strand break repair pathway choice in G2 phase. EMBO J. 30, 1079–1092 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Mimitou, E.P. & Symington, L.S. Ku prevents Exo1 and Sgs1-dependent resection of DNA ends in the absence of a functional MRX complex or Sae2. EMBO J. 29, 3358–3369 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Gravel, S., Chapman, J.R., Magill, C. & Jackson, S.P. DNA helicases Sgs1 and BLM promote DNA double-strand break resection. Genes Dev. 22, 2767–2772 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Chung, W.H., Zhu, Z., Papusha, A., Malkova, A. & Ira, G. Defective resection at DNA double-strand breaks leads to de novo telomere formation and enhances gene targeting. PLoS Genet. 6, e1000948 (2010).

    PubMed  PubMed Central  Google Scholar 

  57. Chen, C. & Kolodner, R.D. Gross chromosomal rearrangements in Saccharomyces cerevisiae replication and recombination defective mutants. Nat. Genet. 23, 81–85 (1999).

    CAS  PubMed  Google Scholar 

  58. Chen, C., Umezu, K. & Kolodner, R.D. Chromosomal rearrangements occur in S. cerevisiae rfa1 mutator mutants due to mutagenic lesions processed by double-strand–break repair. Mol. Cell 2, 9–22 (1998).

    CAS  PubMed  Google Scholar 

  59. Wang, Y. et al. Mutation in Rpa1 results in defective DNA double-strand break repair, chromosomal instability and cancer in mice. Nat. Genet. 37, 750–755 (2005).

    CAS  PubMed  Google Scholar 

  60. Foster, S.S., Balestrini, A. & Petrini, J.H. Functional interplay of the Mre11 nuclease and Ku in the response to replication-associated DNA damage. Mol. Cell. Biol. 31, 4379–4389 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  61. Langston, L.D. & Symington, L.S. Gene targeting in yeast is initiated by two independent strand invasions. Proc. Natl. Acad. Sci. USA 101, 15392–15397 (2004).

    CAS  PubMed  Google Scholar 

  62. Decottignies, A. Microhomology-mediated end joining in fission yeast is repressed by pku70 and relies on genes involved in homologous recombination. Genetics 176, 1403–1415 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  63. Davis, A.P. & Symington, L.S. The yeast recombinational repair protein Rad59 interacts with Rad52 and stimulates single-strand annealing. Genetics 159, 515–525 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  64. Greene, E.C., Wind, S., Fazio, T., Gorman, J. & Visnapuu, M.L. DNA curtains for high-throughput single-molecule optical imaging. Methods Enzymol. 472, 293–315 (2010).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank R. Kolodner (Ludwig Institute for Cancer Research) and R. Rothstein (Columbia University) for the gifts of yeast strains and plasmids, C. Mott for strain construction and W.K. Holloman and members of the Symington lab for comments on the manuscript. This study was supported by grants from the US National Institutes of Health (R01 GM041784 (L.S.S.), R01 GM074739 (E.C.G.) and T32 CA009503 (S.K.D.)). This work was partially supported by the Nanoscale Science and Engineering Initiative of the US National Science Foundation under award CHE-0641523 to E.C.G. and by the New York State Office of Science, Technology and Academic Research (NYSTAR). E.C.G. is supported as an Early Career Scientist with the Howard Hughes Medical Institute.

Author information

Authors and Affiliations

  1. Department of Microbiology and Immunology, Columbia University College of Physicians and Surgeons, New York, New York, USA

    Sarah K Deng, Mariana Justino de Almeida & Lorraine S Symington

  2. Department of Biochemistry and Biophysics, Columbia University College of Physicians and Surgeons, New York, New York, USA

    Bryan Gibb & Eric C Greene

  3. Howard Hughes Medical Institute, Columbia University College of Physicians and Surgeons, New York, New York, USA

    Eric C Greene

Authors
  1. Sarah K Deng
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Bryan Gibb
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mariana Justino de Almeida
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Eric C Greene
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Lorraine S Symington
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

S.K.D., B.G., E.C.G. and L.S.S. designed experiments and wrote the paper. The experiments shown in Figures 1 and 2 and Supplementary Figures 1,2,3,4 were carried out by S.K.D., those shown in Figure 3 were carried out by S.K.D. and B.G., and those shown in Figure 4 were carried out by S.K.D. and M.J.d.A.

Corresponding author

Correspondence to Lorraine S Symington.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 I-SceI cut site and repair sequences.

a, Sequence of Inverted I-SceI site and 12 bp microhomology b, I-SceI cut site with resulting overhangs c, Main classes of NHEJ events d, NHEJ events that yield an Ade+ phenotype. Deletion of any one of the five indicated nucleotides or GATA (4 consecutive nucleotides) restores the ADE2 reading frame to yield Ade+ NHEJ events e, MMEJ using 12 bp microhomology.

Supplementary Figure 2 MMEJ is Ku-independent and partially Sae2 dependent.

a, Linear substrate for blunt end joining (0 MH) or with a direct repeat (8-16 bp) to promote repair by MMEJ. b, Graph of end joining frequencies of the indicated strains; transformations were performed in triplicate and error bars show s.d. c, Graph showing the fraction of precise blunt end joining as determined by sequencing at least 16 independently derived Trp+ clones of the indicated genotypes. d, Graph showing the percent of circularization events that utilize the 12 bp MH as determined by sequencing 15-27 clones.

Supplementary Figure 3 The steady-state protein level of rfa1 mutants.

Western blot of extracts prepared from the indicated strains probed with anti-Rfa1 or anti-α tubulin antibodies (cropped version shown in Figure 2b).

Supplementary Figure 4 End resection and strand invasion in the rfa1 mutants.

a, Schematic representation of the band disappearance resection assay. Loss of the 0.7 and 2.6 kb away fragments indicate resection has proceeded beyond the Styl (S) and XbaI (X) restriction sites rendering them single-stranded and resistant to digestion. The red lines indicate the location of probes used to detect the 0.7 kb and 2.6 kb distal restriction fragments. b, Southern blot of StyI and XbaI digested genomic DNA isolated from wild type, rfa1-D228Y, rfa1-t33, and rfa1-t48 cells. c, Quantification of resection 0.7 kb and 2.6 kb away from the HO cut site. d, Schematic for the mating-type switching assay: the 0.9 kb StyI MATa fragment is cleaved upon HO induction to yield a 0.7 kb cut fragment. The 0.7 kb fragment disappears as resection proceeds and the break is repaired resulting in a 1.8 kb MATα fragment. e, Representative Southern blot of StyI-digested genomic DNA from wild type and rfa1-D228Y cells at different times after HO induction.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4, and Supplementary Tables 1 and 2 (PDF 1219 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Deng, S., Gibb, B., de Almeida, M. et al. RPA antagonizes microhomology-mediated repair of DNA double-strand breaks. Nat Struct Mol Biol 21, 405–412 (2014). https://doi.org/10.1038/nsmb.2786

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.2786

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing