During DNA repair by homologous recombination (HR), DNA synthesis copies information from a template DNA molecule. Multiple DNA polymerases have been implicated in repair-specific DNA synthesis1,2,3, but it has remained unclear whether a DNA helicase is involved in this reaction. A good candidate DNA helicase is Pif1, an evolutionarily conserved helicase in Saccharomyces cerevisiae important for break-induced replication (BIR)4 as well as HR-dependent telomere maintenance in the absence of telomerase5 found in 10–15% of all cancers6. Pif1 has a role in DNA synthesis across hard-to-replicate sites7,8 and in lagging-strand synthesis with polymerase δ (Polδ)9,10,11. Here we provide evidence that Pif1 stimulates DNA synthesis during BIR and crossover recombination. The initial steps of BIR occur normally in Pif1-deficient cells, but Polδ recruitment and DNA synthesis are decreased, resulting in premature resolution of DNA intermediates into half-crossovers. Purified Pif1 protein strongly stimulates Polδ-mediated DNA synthesis from a D-loop made by the Rad51 recombinase. Notably, Pif1 liberates the newly synthesized strand to prevent the accumulation of topological constraint and to facilitate extensive DNA synthesis via the establishment of a migrating D-loop structure. Our results uncover a novel function of Pif1 and provide insights into the mechanism of HR.
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Maloisel, L., Fabre, F. & Gangloff, S. DNA polymerase δ is preferentially recruited during homologous recombination to promote heteroduplex DNA extension. Mol. Cell. Biol. 28, 1373–1382 (2008)
Wang, X. et al. Role of DNA replication proteins in double-strand break-induced recombination in Saccharomyces cerevisiae. Mol. Cell. Biol. 24, 6891–6899 (2004)
Li, X., Stith, C. M., Burgers, P. M. & Heyer, W. D. PCNA is required for initiation of recombination-associated DNA synthesis by DNA polymerase δ. Mol. Cell 36, 704–713 (2009)
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)
Dewar, J. M. & Lydall, D. Pif1- and Exo1-dependent nucleases coordinate checkpoint activation following telomere uncapping. EMBO J. 29, 4020–4034 (2010)
Cesare, A. J. & Reddel, R. R. Alternative lengthening of telomeres: models, mechanisms and implications. Nature Rev. Genet. 11, 319–330 (2010)
Paeschke, K., Capra, J. A. & Zakian, V. A. DNA replication through G-Quadruplex motifs is promoted by the Saccharomyces cerevisiae Pif1 DNA helicase. Cell 145, 678–691 (2011)
Sabouri, N., McDonald, K. R., Webb, C. J., Cristea, I. M. & Zakian, V. A. DNA replication through hard-to-replicate sites, including both highly transcribed RNA Pol II and Pol III genes, requires the S. pombe Pfh1 helicase. Genes Dev. 26, 581–593 (2012)
Budd, M. E., Reis, C. C., Smith, S., Myung, K. & Campbell, J. L. Evidence suggesting that Pif1 helicase functions in DNA replication with the Dna2 helicase/nuclease and DNA polymerase δ. Mol. Cell. Biol. 26, 2490–2500 (2006)
Pike, J. E., Burgers, P. M., Campbell, J. L. & Bambara, R. A. Pif1 helicase lengthens some Okazaki fragment flaps necessitating Dna2 nuclease/helicase action in the two-nuclease processing pathway. J. Biol. Chem. 284, 25170–25180 (2009)
Rossi, M. L. et al. Pif1 helicase directs eukaryotic Okazaki fragments toward the two-nuclease cleavage pathway for primer removal. J. Biol. Chem. 283, 27483–27493 (2008)
Deem, A. et al. Defective break-induced replication leads to half-crossovers in Saccharomyces cerevisiae. Genetics 179, 1845–1860 (2008)
Schulz, V. P. & Zakian, V. A. The saccharomyces PIF1 DNA helicase inhibits telomere elongation and de novo telomere formation. Cell 76, 145–155 (1994)
Lydeard, J. R., Jain, S., Yamaguchi, M. & Haber, J. E. Break-induced replication and telomerase-independent telomere maintenance require Pol32. Nature 448, 820–823 (2007)
Ho, C. K., Mazon, G., Lam, A. F. & Symington, L. S. Mus81 and Yen1 promote reciprocal exchange during mitotic recombination to maintain genome integrity in budding yeast. Mol. Cell 40, 988–1000 (2010)
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)
Prakash, R. et al. Yeast Mph1 helicase dissociates Rad51-made D-loops: implications for crossover control in mitotic recombination. Genes Dev. 23, 67–79 (2009)
Petukhova, G., Stratton, S. & Sung, P. Catalysis of homologous DNA pairing by yeast Rad51 and Rad54 proteins. Nature 393, 91–94 (1998)
Van Komen, S., Petukhova, G., Sigurdsson, S. & Sung, P. Functional cross-talk among Rad51, Rad54, and replication protein A in heteroduplex DNA joint formation. J. Biol. Chem. 277, 43578–43587 (2002)
Bochman, M. L., Sabouri, N. & Zakian, V. A. Unwinding the functions of the Pif1 family helicases. DNA Repair 9, 237–249 (2010)
Sugiyama, T., Zaitseva, E. M. & Kowalczykowski, S. C. A single-stranded DNA-binding protein is needed for efficient presynaptic complex formation by the Saccharomyces cerevisiae Rad51 protein. J. Biol. Chem. 272, 7940–7945 (1997)
Boule, J. B. & Zakian, V. A. The yeast Pif1p DNA helicase preferentially unwinds RNA DNA substrates. Nucleic Acids Res. 35, 5809–5818 (2007)
Sebesta, M., Burkovics, P., Haracska, L. & Krejci, L. Reconstitution of DNA repair synthesis in vitro and the role of polymerase and helicase activities. DNA Repair 10, 567–576 (2011)
Smith, C. E., Lam, A. F. & Symington, L. S. Aberrant double-strand break repair resulting in half crossovers in mutants defective for Rad51 or the DNA polymerase δ complex. Mol. Cell. Biol. 29, 1432–1441 (2009)
Burgers, P. M. & Gerik, K. J. Structure and processivity of two forms of Saccharomyces cerevisiae DNA polymerase δ. J. Biol. Chem. 273, 19756–19762 (1998)
Formosa, T. & Alberts, B. M. DNA synthesis dependent on genetic recombination: characterization of a reaction catalyzed by purified bacteriophage T4 proteins. Cell 47, 793–806 (1986)
Griffith, J. D. & Christiansen, G. Electron microscope visualization of chromatin and other DNA-protein complexes. Annu. Rev. Biophys. Bioeng. 7, 19–35 (1978)
Ferguson, D. O. & Holloman, W. K. Recombinational repair of gaps in DNA is asymmetric in Ustilago maydis and can be explained by a migrating D-loop model. Proc. Natl Acad. Sci. USA 93, 5419–5424 (1996)
Hastings, P. J., Lupski, J. R., Rosenberg, S. M. & Ira, G. Mechanisms of change in gene copy number. Nature Rev. Genet. 10, 551–564 (2009)
Malkova, A. & Ira, G. Break-induced replication: functions and molecular mechanism. Curr. Opin. Genet. Dev. 23, 271–279 (2013)
Sugawara, N., Wang, X. & Haber, J. E. In vivo roles of Rad52, Rad54, and Rad55 proteins in Rad51-mediated recombination. Mol. Cell 12, 209–219 (2003)
Janke, R. et al. A truncated DNA-damage-signaling response is activated after DSB formation in the G1 phase of Saccharomyces cerevisiae. Nucleic Acids Res. 38, 2302–2313 (2010)
Busygina, V. et al. Hed1 regulates Rad51-mediated recombination via a novel mechanism. Genes Dev. 22, 786–795 (2008)
Zierhut, C. & Diffley, J. F. Break dosage, cell cycle stage and DNA replication influence DNA double strand break response. EMBO J. 27, 1875–1885 (2008)
Malkova, A., Klein, F., Leung, W. Y. & Haber, J. E. HO endonuclease-induced recombination in yeast meiosis resembles Spo11-induced events. Proc. Natl Acad. Sci. USA 97, 14500–14505 (2000)
Acharya, N., Klassen, R., Johnson, R. E., Prakash, L. & Prakash, S. PCNA binding domains in all three subunits of yeast DNA polymerase delta modulate its function in DNA replication. Proc. Natl Acad. Sci. USA 108, 17927–17932 (2011)
Van Komen, S., Macris, M., Sehorn, M. G. & Sung, P. Purification and assays of Saccharomyces cerevisiae homologous recombination proteins. Methods Enzymol. 408, 445–463 (2006)
Raschle, M., Van Komen, S., Chi, P., Ellenberger, T. & Sung, P. Multiple interactions with the Rad51 recombinase govern the homologous recombination function of Rad54. J. Biol. Chem. 279, 51973–51980 (2004)
We thank V. Zakian, K. Labib, W.-D. Heyer, J. Haber, C. Wittenberg, R. Johnson and L. Prakash for the gift of antibodies, strains or plasmids, and D. Camerini-Otero for providing DinG. We are grateful to J. Griffith and S. Wilcox for training in metal shadowing electron microscopy. This work was funded by grants from the US National Institutes of Health (GM080600 to G.I.; ES007061, GM057814, ES015632 to P.S., GM084242 to A.M., T32 GM07526-34 to M.A.W.), National Research Foundation of Korea (NRF-2012R1A1A1009917). Academia Sinica, National Taiwan University, and the National Science Council of Taiwan.
The authors declare no competing financial interests.
Extended data figures and tables
Extended Data Figure 1 Analysis of BIR and conversion tracts in pif1Δ mutants and crossover frequency in polymerases mutants.
a, Southern blot analysis of BIR product formation and template chromosome maintenance. Chromosomes were separated by pulsed-field gel electrophoresis and a DNA probe specific for either ADE1 or ADE3 was used. Quantification is shown in Fig. 1c. b, c, Analysis of DSB repair outcomes in the BIR assay of the indicated mutants. d, Schematic of allelic recombination between the leu2 alleles (left). Longer conversion tracts associated with conversion of ‘R’ leads to formation of Leu− recombinants, whereas shorter conversion tracts lead to formation of Leu+ recombinants. Quantification of gene conversion events with shorter conversion tracts (Leu+) in wild-type (WT) and pif1Δ cells is shown. The difference between wild-type and pif1Δ cells is statistically significant, P < 0.0001. e, Southern blot analysis of gene conversion with and without crossing over in the indicated strains using the ectopic recombination assay shown in Fig. 2a. Quantification of crossover product in the indicated mutants compared to wild type that is set to 1. Plotted are the mean values ± s.d. n = 3.
a, Illustration of the half-crossover pathway where the part of the template chromosome distal to the initial invasion site is fused to the broken chromosome, with the remainder of the template chromosome either becoming stabilized (examples shown in Fig. 2b) or lost (as shown in Fig. 2c). b, Analysis of recombination products from Ade+ NATR Leu− colonies. Examples are shown where rearrangements of the template chromosome are indicated by an asterisk. c, Analysis of half-crossover recombination products from Ade− NATR Leu− colonies. d, Analysis of rare NATS Ade+ colonies.
a, Schematic of the ectopic BIR assay. b, Southern blot analysis of ectopic BIR kinetics in wild-type and pif1Δ cells. A probe specific for the MCH2 gene located at the end of chromosome XI was used in the analysis. c, Quantification of ectopic BIR repair (CanS colonies) in wild-type and pif1Δ cells. Plotted are the mean values ± s.d. n = 3. d, e, CHEF analysis of rare products from canavanine-sensitive colonies in pif1Δ (d) and wild-type cells (e). Examples where synthesis is initiated but not finished are indicated by an asterisk. In these cases, a functional CAN1 gene is formed but synthesis is abandoned resulting in shorter products. Red circles indicate major rearrangement of chromosome V or template chromosome XI.
Extended Data Figure 4 Analysis of Pif1’s role in the initial steps of BIR and of Pif1 recruitment at the DSB and template.
a, b, Analysis of initial strand invasion in wild-type and pif1Δ cells. a, Enrichment of Rad51 at the DSB site and template by ChIP analysis. b, Kinetics of removal of the non-homologous Ya tail by qPCR analysis in wild-type and pif1-m2 strains compared to the control strains rad51Δ and rad54Δ that are defective in strand invasion. c, Enrichment of Pif1 at the DSB and template by ChIP analysis in wild-type, pol32 and rad52 cells. The regions amplified by qPCR are indicated. d, Control ChIP experiments in the PIF1-13×Myc strain where crosslinking was omitted and in a strain where the Myc tag was absent. a–d, plotted are the mean values ± s.d. n = 3.
Extended Data Figure 5 Quality analyses of proteins, protein requirements for DNA extension, effect on Pif1 on D-loop stability, and time-course analysis of DNA extension.
a, Purified Pif1 and pif1(K264A) were analysed by SDS–PAGE and staining with Coomassie blue. b, The plasmid DNA in all the lanes was pBluescript SK replicative form I (RFI). DNA synthesis reactions were performed with 13, 27 and 40 nM Pif1 and the reaction mixtures (lanes 1–8) from the 8-min time point were incubated at 95 °C for 2 min to disrupt the D-loop, followed by native gel electrophoresis and staining with ethidium bromide. Various other DNA forms (lane 9, plasmid DNA alone; lane 10, plasmid DNA linearized with XhoI; lane 11, plasmid DNA relaxed by calf thymus topoisomerase I; lane 12, plasmid DNA relaxed by E. coli topoisomerase I; lane 13, plasmid DNA digested with DNase I) are shown. c, DNA synthesis reactions by Polδ in conjunction with Pif1 (40 nM Pif1 and 8-min incubation) with the omission of one or more of the protein factors or dNTPs, as indicated. The reaction products were analysed in a native gel (top) or denaturing gel (bottom). Note that a substantial portion of the D-loop was dissociated by Pif1 in the absence of PCNA, RFC, Polδ, or dNTPs (lanes 5, 7, 9 and 19). d, Time course of DNA synthesis by Polδ in conjunction with Pif1 (40 nM Pif1). The reaction products were analysed in a native gel (top) or a denaturing gel (bottom).
a, DNA synthesis from a deproteinized D-loop by Polδ in conjunction with Pif1 (8, 16 and 24 nM) was examined. Pif1 was at 24 nM in lane 6. The reaction products were analysed in a native gel (left) or denaturing gel (right). b, After the D-loop reaction had proceeded for 2 min, Rad54, which is highly heat labile19, was inactivated by incubation at 42 °C for 20 min. DNA extension reaction and analysis were then performed by adding RPA, RFC, PCNA, Polδ and Pif1 (40 nM Pif1 and 8-min incubation). The reaction products were analysed in a native gel (left) or denaturing gel (right). The inactivation of Rad54 was verified by examining the ATPase activity of Rad54, which decreased by ∼95% compared to the unheated control (data not shown).
a, DNA synthesis reactions were conducted with Polδ and Pif1, Mph1, Rrm3, or DinG (13, 27, 40, 120 nM). The reaction products from the 8-min time point were resolved in a native (top) or denaturing gel (bottom) (lane 1, no protein control; lane 2, D-loop formed by Rad51–Rad54; lanes 3–20, D-loop extended with Polδ and the indicated helicase). b, E. coli DNA polymerase I Klenow fragment (100 nM, from NEB) was tested for DNA extension with Pif1 (13, 27, 40 nM) with or without PCNA (200 nM) and RFC (200 nM). The reaction products from the 8-min time point were analysed in a native gel.
Extended Data Figure 8 Requirement for the Polδ subunit Pol32 in DNA extension, and interaction of Pif1 with PCNA.
a, Purified Polδ (Flag–Pol3, GST–Pol31, Pol32), Polδ* (Flag–Pol3, GST–Pol31) and MBP–Pol32 were analysed by SDS–PAGE and staining with Coomassie blue. b, Pull-down assay to examine Pol32–Polδ* interaction. c, DNA synthesis was performed with Polδ or Polδ* (20 or 40 nM) with Pif1 (40 nM). In lanes 10–13, Polδ* and Pol32 (125 nM) were pre-incubated on ice for 10 min before use. The reaction products from the 8-min time point were resolved in a native gel (left) or denaturing gel (right). d, Pull-down reactions of 6×His–Pif1 and PCNA (left), Polδ (Flag–Pol3, GST–Pol31, Pol32) and PCNA, Polδ and Pif1 (right). E, SDS eluate; S, supernatant; W, wash.
a, DNA synthesis products, initiated by Polδ for 4 min, and then continued with Pif1 (13, 27, 40 nM) with and without calf thymus topoisomerase I (0.4 U μl−1) for 8 min. The reaction mixtures were resolved in a native gel (top) or denaturing gel (middle). Lanes 1 and 2 contained DNA substrates only and lanes 3–12 contained D-loop made by Rad51–Rad54. An overexposed image and the scan of lanes 5 and 6 to highlight the effect of topoisomerase when Pif1 was absent are shown (bottom). b, Two-dimensional gel analysis of the extension products. The reaction products, prepared as in lanes 5, 6, 11 and 12 of panel a, were subject to two-dimensional gel analysis.
Extended Data Figure 10 Measurement of ssDNA intermediates formed during BIR and analysis of BIR efficiency in the absence of Psf2 and Mcm4.
a, Schematic of the assay. b, Measurement of the relative increase of ssDNA at the indicated time after DSB induction compared to the amount of ssDNA in logarithmically growing cells (t = 0). Measurement of ssDNA intermediate 10 and 40 kb from the site of strand invasion at the template chromosome and at a control locus on chromosome V which does not participate in recombination. Plotted are the mean values ± s.d. n = 3. c, An analysis of the growth of cells harbouring temperature-sensitive degron alleles of td-mcm4 and td-psf2. Both strains are inviable at 37 °C even without overexpression of the ubiquitin ligase Ubr1. d, Western blot analysis of td-Mcm4 and td-Psf2 protein degradation. e, Southern blot analysis of the BIR assay in cells with conditional depletion of td-Mcm4 or td-Psf2. Quantification of the Southern blots is shown in Fig. 2f.
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Wilson, M., Kwon, Y., Xu, Y. et al. Pif1 helicase and Polδ promote recombination-coupled DNA synthesis via bubble migration. Nature 502, 393–396 (2013). https://doi.org/10.1038/nature12585
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