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.

Visualization of recombination-mediated damage bypass by template switching

Abstract

Template switching (TS) mediates damage bypass via a recombination-related mechanism involving PCNA polyubiquitination and polymerase δ–dependent DNA synthesis. Using two-dimensional gel electrophoresis and EM, here we characterize TS intermediates arising in Saccharomyces cerevisiae at a defined chromosome locus, identifying five major families of intermediates. Single-stranded DNA gaps of 150–200 nt, and not DNA ends, initiate TS by strand invasion. This causes reannealing of the parental strands and exposure of the nondamaged newly synthesized chromatid, which serves as a replication template for the other blocked nascent strand. Structures resembling double Holliday junctions, postulated to be central double-strand break–repair intermediates but so far visualized only in meiosis, mediate late stages of TS before being processed to hemicatenanes. Our results reveal the DNA transitions accounting for recombination-mediated DNA-damage tolerance in mitotic cells and replication under conditions of genotoxic stress.

This is a preview of subscription content

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: TS intermediates formed on YLpFAT7.1 minichromosomes.
Figure 2: X molecule–intermediate isolation and purification.
Figure 3: Representative EM pictures of F1 and F2 families.
Figure 4: Representative EM pictures of F3, F4 and F5 families.
Figure 5: Branch migration assays and asymmetric families of JMs.
Figure 6: Observation of F3 molecules by denaturing spreading and biochemical identification of paranemic pairing.
Figure 7: Hypothetical model of TS.

References

  1. Branzei, D. & Foiani, M. Maintaining genome stability at the replication fork. Nat. Rev. Mol. Cell Biol. 11, 208–219 (2010).

    CAS  Article  Google Scholar 

  2. Weinert, T., Kaochar, S., Jones, H., Paek, A. & Clark, A.J. The replication fork's five degrees of freedom, their failure and genome rearrangements. Curr. Opin. Cell Biol. 21, 778–784 (2009).

    CAS  Article  Google Scholar 

  3. Sale, J.E. Competition, collaboration and coordination: determining how cells bypass DNA damage. J. Cell Sci. 125, 1633–1643 (2012).

    CAS  Article  Google Scholar 

  4. Zhang, H. & Lawrence, C.W. The error-free component of the RAD6/RAD18 DNA damage tolerance pathway of budding yeast employs sister-strand recombination. Proc. Natl. Acad. Sci. USA 102, 15954–15959 (2005).

    CAS  Article  Google Scholar 

  5. Prakash, L. Characterization of postreplication repair in Saccharomyces cerevisiae and effects of rad6, rad18, rev3 and rad52 mutations. Mol. Gen. Genet. 184, 471–478 (1981).

    CAS  Article  Google Scholar 

  6. Lehmann, A.R. Postreplication repair of DNA in ultraviolet-irradiated mammalian cells. J. Mol. Biol. 66, 319–337 (1972).

    CAS  Article  Google Scholar 

  7. Branzei, D., Vanoli, F. & Foiani, M. SUMOylation regulates Rad18-mediated template switch. Nature 456, 915–920 (2008).

    CAS  Article  Google Scholar 

  8. Hoege, C., Pfander, B., Moldovan, G.L., Pyrowolakis, G. & Jentsch, S. RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO. Nature 419, 135–141 (2002).

    CAS  Article  Google Scholar 

  9. Stelter, P. & Ulrich, H.D. Control of spontaneous and damage-induced mutagenesis by SUMO and ubiquitin conjugation. Nature 425, 188–191 (2003).

    CAS  Article  Google Scholar 

  10. Pfander, B., Moldovan, G.L., Sacher, M., Hoege, C. & Jentsch, S. SUMO-modified PCNA recruits Srs2 to prevent recombination during S phase. Nature 436, 428–433 (2005).

    CAS  Article  Google Scholar 

  11. Papouli, E. et al. Crosstalk between SUMO and ubiquitin on PCNA is mediated by recruitment of the helicase Srs2p. Mol. Cell 19, 123–133 (2005).

    CAS  Article  Google Scholar 

  12. Minca, E.C. & Kowalski, D. Multiple Rad5 activities mediate sister chromatid recombination to bypass DNA damage at stalled replication forks. Mol. Cell 38, 649–661 (2010).

    CAS  Article  Google Scholar 

  13. Haracska, L., Torres-Ramos, C.A., Johnson, R.E., Prakash, S. & Prakash, L. 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 (2004).

    CAS  Article  Google Scholar 

  14. Lopes, M., Foiani, M. & Sogo, J.M. Multiple mechanisms control chromosome integrity after replication fork uncoupling and restart at irreparable UV lesions. Mol. Cell 21, 15–27 (2006).

    CAS  Article  Google Scholar 

  15. Heller, R.C. & Marians, K.J. Replication fork reactivation downstream of a blocked nascent leading strand. Nature 439, 557–562 (2006).

    CAS  Article  Google Scholar 

  16. Torres-Ramos, C.A., Prakash, S. & Prakash, L. Requirement of RAD5 and MMS2 for postreplication repair of UV-damaged DNA in Saccharomyces cerevisiae. Mol. Cell. Biol. 22, 2419–2426 (2002).

    CAS  Article  Google Scholar 

  17. Vanoli, F., Fumasoni, M., Szakal, B., Maloisel, L. & Branzei, D. Replication and recombination factors contributing to recombination-dependent bypass of DNA lesions by template switch. PLoS Genet. 6, e1001205 (2010).

    Article  Google Scholar 

  18. Torres-Ramos, C.A., Prakash, S. & Prakash, L. Requirement of yeast DNA polymerase δ in post-replicational repair of UV-damaged DNA. J. Biol. Chem. 272, 25445–25448 (1997).

    CAS  Article  Google Scholar 

  19. Karras, G.I. & Jentsch, S. The RAD6 DNA damage tolerance pathway operates uncoupled from the replication fork and is functional beyond S phase. Cell 141, 255–267 (2010).

    CAS  Article  Google Scholar 

  20. Liberi, G. et al. 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 (2005).

    CAS  Article  Google Scholar 

  21. Ashton, T.M., Mankouri, H.W., Heidenblut, A., McHugh, P.J. & Hickson, I.D. Pathways for Holliday junction processing during homologous recombination in Saccharomyces cerevisiae. Mol. Cell. Biol. 31, 1921–1933 (2011).

    CAS  Article  Google Scholar 

  22. Szakal, B. & Branzei, D. Premature Cdk1/Cdc5/Mus81 pathway activation induces aberrant replication and deleterious crossover. EMBO J. 32, 1155–1167 (2013).

    CAS  Article  Google Scholar 

  23. Mankouri, H.W., Ashton, T.M. & Hickson, I.D. Holliday junction-containing DNA structures persist in cells lacking Sgs1 or Top3 following exposure to DNA damage. Proc. Natl. Acad. Sci. USA 108, 4944–4949 (2011).

    CAS  Article  Google Scholar 

  24. Osman, F. & Whitby, M.C. Exploring the roles of Mus81-Eme1/Mms4 at perturbed replication forks. DNA Repair (Amst.) 6, 1004–1017 (2007).

    CAS  Article  Google Scholar 

  25. Sung, P. & Klein, H. Mechanism of homologous recombination: mediators and helicases take on regulatory functions. Nat. Rev. Mol. Cell Biol. 7, 739–750 (2006).

    CAS  Article  Google Scholar 

  26. Branzei, D. Ubiquitin family modifications and template switching. FEBS Lett. 585, 2810–2817 (2011).

    CAS  Article  Google Scholar 

  27. Ciccia, A. et al. Polyubiquitinated PCNA recruits the ZRANB3 translocase to maintain genomic integrity after replication stress. Mol. Cell 47, 396–409 (2012).

    CAS  Article  Google Scholar 

  28. Blastyák, A. et al. Yeast Rad5 protein required for postreplication repair has a DNA helicase activity specific for replication fork regression. Mol. Cell 28, 167–175 (2007).

    Article  Google Scholar 

  29. Burkovics, P., Sebesta, M., Balogh, D., Haracska, L. & Krejci, L. Strand invasion by HLTF as a mechanism for template switch in fork rescue. Nucleic Acids Res. 42, 1711–1720 (2014).

    CAS  Article  Google Scholar 

  30. Glineburg, M.R., Chavez, A., Agrawal, V., Brill, S.J. & Johnson, F.B. Resolution by unassisted Top3 points to template switch recombination intermediates during DNA replication. J. Biol. Chem. 288, 33193–33204 (2013).

    CAS  Article  Google Scholar 

  31. Lehmann, A.R. & Fuchs, R.P. Gaps and forks in DNA replication: rediscovering old models. DNA Repair (Amst.) 5, 1495–1498 (2006).

    CAS  Article  Google Scholar 

  32. Alexandrov, L.B. et al. Signatures of mutational processes in human cancer. Nature 500, 415–421 (2013).

    CAS  Article  Google Scholar 

  33. Tateishi, S., Sakuraba, Y., Masuyama, S., Inoue, H. & Yamaizumi, M. Dysfunction of human Rad18 results in defective postreplication repair and hypersensitivity to multiple mutagens. Proc. Natl. Acad. Sci. USA 97, 7927–7932 (2000).

    CAS  Article  Google Scholar 

  34. Hishida, T., Kubota, Y., Carr, A.M. & Iwasaki, H. RAD6–RAD18–RAD5-pathway-dependent tolerance to chronic low-dose ultraviolet light. Nature 457, 612–615 (2009).

    CAS  Article  Google Scholar 

  35. Daigaku, Y., Davies, A.A. & Ulrich, H.D. Ubiquitin-dependent DNA damage bypass is separable from genome replication. Nature 465, 951–955 (2010).

    CAS  Article  Google Scholar 

  36. Alvaro, D., Lisby, M. & Rothstein, R. Genome-wide analysis of Rad52 foci reveals diverse mechanisms impacting recombination. PLoS Genet. 3, e228 (2007).

    Article  Google Scholar 

  37. Mozlin, A.M., Fung, C.W. & Symington, L.S. Role of the Saccharomyces cerevisiae Rad51 paralogs in sister chromatid recombination. Genetics 178, 113–126 (2008).

    CAS  Article  Google Scholar 

  38. Fabre, F., Chan, A., Heyer, W.D. & Gangloff, S. Alternate pathways involving Sgs1/Top3, Mus81/ Mms4, and Srs2 prevent formation of toxic recombination intermediates from single-stranded gaps created by DNA replication. Proc. Natl. Acad. Sci. USA 99, 16887–16892 (2002).

    CAS  Article  Google Scholar 

  39. Runge, K.W. & Zakian, V.A. Introduction of extra telomeric DNA sequences into Saccharomyces cerevisiae results in telomere elongation. Mol. Cell. Biol. 9, 1488–1497 (1989).

    CAS  Article  Google Scholar 

  40. Bzymek, M., Thayer, N.H., Oh, S.D., Kleckner, N. & Hunter, N. Double Holliday junctions are intermediates of DNA break repair. Nature 464, 937–941 (2010).

    CAS  Article  Google Scholar 

  41. Neelsen, K.J., Chaudhuri, A.R., Follonier, C., Herrador, R. & Lopes, M. Visualization and interpretation of eukaryotic DNA replication intermediates in vivo by electron microscopy. Methods Mol. Biol. 1094, 177–208 (2014).

    CAS  Article  Google Scholar 

  42. Cromie, G.A. et al. Single Holliday junctions are intermediates of meiotic recombination. Cell 127, 1167–1178 (2006).

    CAS  Article  Google Scholar 

  43. Liberi, G. et al. Methods to study replication fork collapse in budding yeast. Methods Enzymol. 409, 442–462 (2006).

    CAS  Article  Google Scholar 

  44. Lopes, M., Cotta-Ramusino, C., Liberi, G. & Foiani, M. Branch migrating sister chromatid junctions form at replication origins through Rad51/Rad52-independent mechanisms. Mol. Cell 12, 1499–1510 (2003).

    CAS  Article  Google Scholar 

  45. Allers, T. & Lichten, M. A method for preparing genomic DNA that restrains branch migration of Holliday junctions. Nucleic Acids Res. 28, e6 (2000).

    CAS  Article  Google Scholar 

  46. Joo, C., McKinney, S.A., Lilley, D.M. & Ha, T. Exploring rare conformational species and ionic effects in DNA Holliday junctions using single-molecule spectroscopy. J. Mol. Biol. 341, 739–751 (2004).

    CAS  Article  Google Scholar 

  47. Cejka, P., Plank, J.L., Bachrati, C.Z., Hickson, I.D. & Kowalczykowski, S.C. Rmi1 stimulates decatenation of double Holliday junctions during dissolution by Sgs1–Top3. Nat. Struct. Mol. Biol. 17, 1377–1382 (2010).

    CAS  Article  Google Scholar 

  48. Gonzalez-Huici, V. et al. DNA bending facilitates the error-free DNA damage tolerance pathway and upholds genome integrity. EMBO J. 33, 327–340 (2014).

    CAS  Article  Google Scholar 

  49. Follonier, C., Oehler, J., Herrador, R. & Lopes, M. Friedreich's ataxia–associated GAA repeats induce replication-fork reversal and unusual molecular junctions. Nat. Struct. Mol. Biol. 20, 486–494 (2013).

    CAS  Article  Google Scholar 

  50. Sogo, J.M., Lopes, M. & Foiani, M. Fork reversal and ssDNA accumulation at stalled replication forks owing to checkpoint defects. Science 297, 599–602 (2002).

    CAS  Article  Google Scholar 

  51. Szostak, J.W., Orr-Weaver, T.L., Rothstein, R.J. & Stahl, F.W. The double-strand-break repair model for recombination. Cell 33, 25–35 (1983).

    CAS  Article  Google Scholar 

  52. Karras, G.I. et al. Noncanonical role of the 9-1-1 clamp in the error-free DNA damage tolerance pathway. Mol. Cell 49, 536–546 (2013).

    CAS  Article  Google Scholar 

  53. Wu, L. & Hickson, I.D. The Bloom's syndrome helicase suppresses crossing over during homologous recombination. Nature 426, 870–874 (2003).

    CAS  Article  Google Scholar 

  54. Ira, G., Malkova, A., Liberi, G., Foiani, M. & Haber, J.E. Srs2 and Sgs1-Top3 suppress crossovers during double-strand break repair in yeast. Cell 115, 401–411 (2003).

    CAS  Article  Google Scholar 

  55. Bugreev, D.V., Yu, X., Egelman, E.H. & Mazin, A.V. Novel pro- and anti-recombination activities of the Bloom's syndrome helicase. Genes Dev. 21, 3085–3094 (2007).

    CAS  Article  Google Scholar 

  56. Hu, Y. et al. RECQL5/Recql5 helicase regulates homologous recombination and suppresses tumor formation via disruption of Rad51 presynaptic filaments. Genes Dev. 21, 3073–3084 (2007).

    CAS  Article  Google Scholar 

  57. Robert, T., Dervins, D., Fabre, F. & Gangloff, S. Mrc1 and Srs2 are major actors in the regulation of spontaneous crossover. EMBO J. 25, 2837–2846 (2006).

    CAS  Article  Google Scholar 

  58. Gangloff, S., McDonald, J.P., Bendixen, C., Arthur, L. & Rothstein, R. The yeast type I topoisomerase Top3 interacts with Sgs1, a DNA helicase homolog: a potential eukaryotic reverse gyrase. Mol. Cell. Biol. 14, 8391–8398 (1994).

    CAS  Article  Google Scholar 

  59. Branzei, D. et al. Ubc9- and Mms21-mediated sumoylation counteracts recombinogenic events at damaged replication forks. Cell 127, 509–522 (2006).

    CAS  Article  Google Scholar 

  60. Sollier, J. et al. The Saccharomyces cerevisiae Esc2 and Smc5–6 proteins promote sister chromatid junction-mediated intra-S repair. Mol. Biol. Cell 20, 1671–1682 (2009).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

This work was supported by European Research Council (ERC), Associazione Italiana per la Ricerca sul Cancro (AIRC) and Fondazione Telethon grants to D.B., Swiss National Science Foundation grants PP00P3_135292 and 31003A_146924 to K.Z., C.F. and M.L., and AIRC and Fondazione Telethon grants to M.F. We thank the Center for Microscopy and Image Analysis of the University of Zürich for technical assistance with the EM experiments, I. Psakhye for critical reading of the manuscript, W. Carotenuto at IFOM for initial assistance in drawing the model, members of our laboratories for helpful discussions and FIRC for various support.

Author information

Authors and Affiliations

Authors

Contributions

M.G. designed and executed the experiments, acquired the EM images, analyzed the data and made the figures. K.Z. and C.F. acquired a subset of EM images and helped with EM data analysis. M.F. conceived the project and discussed the results. M.L. conceived the project, supervised the EM part, analyzed the EM data and commented on the manuscript. D.B. conceived and supervised the project, designed the experiments, analyzed the data and wrote the paper.

Corresponding author

Correspondence to Dana Branzei.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Features of the minichromosome YLpFAT7.1 and minichromosome-derived template switch–intermediate families.

(a) Schematic representation of the minichromosome YLpFAT7.1 with the location of the leu2d probe. (b) Undigested or BglII digested genomic DNA samples from wild-type (wt, CY11340), sgs1Δ (CY11357), rad51Δ (CY12388), sgs1Δ rad51Δ (CY12390) strains carrying the minichromosome YLpFAT7.1 were analyzed by gel electrophoresis. The minichromosome was visualized by Southern blotting using the leu2d probe. (c) Genomic DNA from wt (CY11340) and sgs1Δ (CY11357) strains was digested with a restriction enzymes mix containing PacI, XhoI, SgrAI, BssHII, SpeI, BclI and PmlI, and an aliquot of the digestion mix was examined on a first dimension gel (0.35% agarose TBE 1X). After ethidium bromide staining, the minichromosome band is visible at the expected molecular weight and the endogenous genomic DNA is cleaved to an average length smaller than 5kb. (d) Number and percentage of X-shaped DNA structures found in the wt and sgs1Δ samples that were assigned as minichromosome-derived structures. (e) Box plot showing the distribution of the ssDNA lengths in F1*-F1-F2 molecules in wt and sgs1Δ cells. Center line, median; box limits, 25th and 75th percentiles; whiskers, 10th and 90th percentiles; dots, outliers; (n), number of samples (molecules) in the data set. The median (M) and average (A) values derived from calculations are displayed. NS, P> 0.05 by t-test with two tails (f) Chart representing the distribution of the JM families in wt and sgs1Δ cells.

Supplementary Figure 2 Representative EM pictures of F1 family of molecules.

(a-c) Total views (left panels) of representative F1 family DNA structures and enlarged views (right panels) of the junction points. Branch sizes until the junction point are reported in kilobases. Two colors, grey and black, in which the length values are displayed, mark the “equal” arms. White arrows mark the junction points and red arrows the ssDNA discontinuities on one branch in immediate proximity to the junction point. Schematic representations of the junction points are shown with a black/grey code to indicate the contributions of the DNA duplexes to the junction and with a black/red code to indicate the ssDNA regions in red and dsDNA regions in black. Scale bars are shown.

Supplementary Figure 3 Representative EM pictures of F2 family of molecules.

(a-c) Total views (left panels) of representative F2 family of DNA structures and enlarged views (right panels) of the junction points. Branch sizes until the junction point are reported in kilobases. Two colors, grey and black, in which the length values are displayed, mark the “equal” arms. White arrows mark the junction points, which have an extended region of homology (double Y-like structures). Black arrows mark the thick DNA filament at the junction point that we infer to be three-stranded for the reasons explained in the main text. Red arrows mark the ssDNA discontinuity on one or two branches of the joint molecule in immediate proximity to the junction point. Black asterisks mark random crosses between DNA filaments. Schematic representations of the junction points are shown with black/grey and black/red codes as in Supplementary Figure 2. Scale bars are shown.

Supplementary Figure 4 Representative EM pictures of F3 family of molecules.

(a-c) Total views (left panels) of representative F3 family of DNA structures and enlarged views (right panels) of the junction points. Branch sizes until the junction point are reported in kilobases. Two colors, grey and black, in which the length values are displayed, mark the “equal” arms. White arrows mark the junction points, which have an extended region of homology (bubble-like structures). Lengths of the dsDNA filaments at the junction points of F3 molecules are reported in white in kilobases. Black asterisks mark random crosses between DNA filaments. Schematic representations of the junction points are shown with black/grey and black/red codes as in Supplementary Figure 2. Scale bars are shown.

Supplementary Figure 5 Representative EM pictures of F3* family of molecules.

(a-c) Total views (left panels) of representative F3* family of DNA structures and enlarged views (right panels) of the junction points. Branch sizes until the junction point are reported in kilobases. Two colors, grey and black, in which the length values are displayed, mark the “equal” arms. White arrows mark the junction points, which have an extended region of homology (bubble-like structures), and red arrows mark the ssDNA discontinuity on one of the dsDNA filaments present at the junction point. Lengths of the DNA filaments at the junction points are reported in white in kilobases. Schematic representations of the junction points are shown with black/grey and black/red codes as in Supplementary Figure 2. Scale bars are shown.

Supplementary Figure 6 Representative EM pictures of F4 family of molecules.

(a-c) Total views (left panels) of representative F4 family of DNA structures and enlarged views (right panels) of the junction points. Branch sizes until the junction point are reported in kilobases. Two colors, grey and black, in which the length values are displayed, mark the “equal” arms. White arrows mark the junction points, which have an extended region of homology (double Y-like structures). Schematic representations of the junction points are shown with black/grey color code as in Supplementary Figure 2. Scale bars are shown.

Supplementary Figure 7 Representative EM pictures of F5 family of molecules.

(a-c) Total views (left panels) of representative F5 family of DNA structures and enlarged views (right panels) of the junction points. Branch sizes until the junction point are reported in kilobases. Two colors, grey and black, in which the length values are displayed, mark the “equal” arms. White arrows mark the junction points and red arrows the ssDNA regions. Schematic representations of the junction points are shown with black/grey and black/red codes as in Supplementary Figure 2. Scale bars are shown.

Supplementary Figure 8 Representative EM pictures of single Holliday junctions and asymmetric F5* and F1* types of molecules.

(a) A representative EM picture of a single HJ-like molecule (sHJ) with a total view of the DNA structure and an enlarged view of the junction point with a schematic representation of the ssDNA regions reported in red is shown. Branch sizes until the junction point are reported in black in kilobases. Schematic representation of the junction point is also shown with a black/grey code to indicate the contributions of two DNA duplexes to the junction. Scale bars are shown. (b-c) Total views of X-shaped DNA structures of F5* family (b) or F1* family (c) and enlarged views of the junction points with schematic representations of the ssDNA regions in red. Branch sizes until the junction point are reported in kilobases. Two colors, blue and black, used to indicate the length values mark the arms belonging to the same DNA duplex involved in the junction. Schematic representations of the junction points are shown with black/grey and black/red codes as in Supplementary Figure 2. Scale bars are shown.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 and Supplementary Table 1 (PDF 14969 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Giannattasio, M., Zwicky, K., Follonier, C. et al. Visualization of recombination-mediated damage bypass by template switching. Nat Struct Mol Biol 21, 884–892 (2014). https://doi.org/10.1038/nsmb.2888

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.2888

Further reading

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