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. 2019 Dec 5;76(5):699-711.e6.
doi: 10.1016/j.molcel.2019.08.017. Epub 2019 Sep 18.

Rad52 Restrains Resection at DNA Double-Strand Break Ends in Yeast

Zhenxin Yan  1 , Chaoyou Xue  2 , Sandeep Kumar  1 , J Brooks Crickard  2 , Yang Yu  1 , Weibin Wang  3 , Nhung Pham  1 , Yuxi Li  4 , Hengyao Niu  4 , Patrick Sung  5 , Eric C Greene  6 , Grzegorz Ira  7
Affiliations

Rad52 Restrains Resection at DNA Double-Strand Break Ends in Yeast

Zhenxin Yan et al. Mol Cell. .

Abstract

Rad52 is a key factor for homologous recombination (HR) in yeast. Rad52 helps assemble Rad51-ssDNA nucleoprotein filaments that catalyze DNA strand exchange, and it mediates single-strand DNA annealing. We find that Rad52 has an even earlier function in HR in restricting DNA double-stranded break ends resection that generates 3' single-stranded DNA (ssDNA) tails. In fission yeast, Exo1 is the primary resection nuclease, with the helicase Rqh1 playing a minor role. We demonstrate that the choice of two extensive resection pathways is regulated by Rad52. In rad52 cells, the resection rate increases from ∼3-5 kb/h up to ∼10-20 kb/h in an Rqh1-dependent manner, while Exo1 becomes dispensable. Budding yeast Rad52 similarly inhibits Sgs1-dependent resection. Single-molecule analysis with purified budding yeast proteins shows that Rad52 competes with Sgs1 for DNA end binding and inhibits Sgs1 translocation along DNA. These results identify a role for Rad52 in limiting ssDNA generated by end resection.

Keywords: DNA repair; Rad52; RecQ helicase; double-strand break; homologous recombination; resection; yeast.

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Conflict of interest statement

DECLARATION OF INTERESTS

The authors declare no competing interests.

Figures

Figure 1.
Figure 1.. Role of MRN, Ctp1, Exo1, and Rqh1 in Initial and Extensive Resection
(A and B) Southern blot analysis of initial resection (0–150 min) (A) and extensive resection (0–12 h) (B) at lys1 locus in wild-type, mre11, and ctp1 cells.(C and D) Southern blot analysis of initial resection (0–150 min) (C) and extensive resection (0–12 h) (D) at lys1 locus in wild-type and exo1, rqh1, or exo1 rqh1 mutant cells. Restriction-enzyme-digested DNA was separated on 0.8% agarose gels. Primers used to prepare DNA probes for Southern blotting and restriction enzymes used to digest genomic DNA are presented in Table S3. Plots show kinetics of resection and error bars denote SD (n = 3). An asterisk in (C) denotes unresected DSB end.
Figure 2.
Figure 2.. Rad52 Negatively Controls Extensive Resection in Fission Yeast
(A and B) Southern blot analysis of resection in wild-type and rad52Δ strains at the (A) lys1 locus or (B) arg1 locus. DNA was separated on 0.8% agarose gels. (C) Southern blot analysis of resection in cells with overexpressed Rad52. Plots show kinetics of resection, and error bars denote SD (n = 3).
Figure 3.
Figure 3.. Role of the C-Terminal Region of Rad52 in Extensive Resection
(A) Schematic showing major Rad52 domains and truncation mutants analyzed. (B) Southern blot analysis of resection in indicated rad52 truncation mutants. (C) Plots showing kinetics of resection in rad52 mutants. Error bars denote SD (n = 3). (D) Analysis of sensitivity to DNA damage of wild-type and rad52 mutant strains. 5-fold serial dilutions were made, and 2 μL was spotted onto yeast extract with supplements (YES) or YES with hydroxyurea (HU), camptothecin (CPT), methyl methanesulfonate (MMS), or phleomycin. (E) ChIP-qPCR analysis of recruitment of RPA (Rfa1 subunit, called Ssb1 in fission yeast) in wild-type and rad52ΔC80 cells at 1 and 58 kb from the DSB end at lys1 locus. Error bars denote SD (n = 3). One-tailed p values were shown (*p < 0.05).
Figure 4.
Figure 4.. Analysis of DNA-Binding Domain of Rad52 in Resection Control
(A) Sequence comparison of yeasts and human Rad52. (B) Schematic of new SSA assay between two ~0.75-kb λ2 repeats inserted at arg1 locus and 21 kb upstream of arg1. (C) Southern blot analysis of SSA in indicated rad52 mutants deficient in DNA binding (rad52-R45A) or C-terminal domain (rad52ΔC80). Plot shows kinetics of SSA product formation. Error bars denote SD (n = 3). (D) Comparison of extensive resection kinetics in wild-type and rad52-R45A mutant cells. Plots show kinetics of resection, and error bars denote SD (n = 3).
Figure 5.
Figure 5.. Rad52 Controls Rqh1-Mediated Resection and Rqh1 Recruitment
(A) Epistasis analysis between Rad52 and enzymes mediating two extensive resection pathways, Exo1 and Rqh1. Southern blot analysis of resection in indicated mutants. Plots show kinetics of resection, and error bars denote SD (n = 3). (B and C) ChIP-qPCR analysis of Rqh1 and Exo1 loading in wild-type and rad52 cells at a DSB at lys1 locus at 1 and 10 kb from DSB ends. Error bars denote SD (Rqh1-FLAG: wild type [WT], n = 7, rad52Δ, n = 8. Exo1-FLAG: n = 3). (B) One-tailed p values were shown (**p < 0.01, ***p < 0.001). (C) One-tailed p values were shown (*p < 0.05).
Figure 6.
Figure 6.. Rad52 Can Block DNA End Binding by Sgs1
(A) Analysis of resection kinetics at DSB at MAT locus in budding yeast wild-type cells and cells that either lack or overexpress Rad52. Genomic DNA was digested with EcoRI; primers used to prepare DNA probes for Southern blotting are presented in Table S4. Corresponding Southern blots are shown in Figures S6A and 6B. Dox, doxycycline. (B) ChIP-qPCR analysis of Sgs1 loading at MAT locus 3 h after DSB induction in wild-type and rad52Δ cells. Error bars denote SD (n = 6). One-tailed p values were shown (**p < 0.01). (C) (Left) Wide-field images of GFP-Sgs1 (green) pre-bound to dsDNA ends after addition of Alexa546-Rad52 (magenta); “T” and “F” refer to the tethered and free DNA ends. (Right) Initial distribution of pre-bound GFP-Sgs1 (N = 102) that was chased by addition of Alexa546-Rad52; error bars represent 95% confidence intervals (CIs), and the blue line represents a Gaussian fit to the data. (D) (Left) Kymograph showing the dissociation of end-bound GFP-Sgs1 (green) after addition of Alexa546-Rad52 (magenta). Arrowheads denote the time point of the Alexa546-Rad52 injection. (Right) Survival probability for end-bound GFP-Sgs1 (±ATP) chased with Rad52 or unlabeled Sgs1, as indicted. Note, that the minus Rad52 data curves are reproduced from Xue et al. (2019) for comparison. (E) (Left) Wide-field images showing dsDNA pre-bound by Alexa546-Rad52 (magenta) and then chased with GFP-Sgs1 (green). (Right) The corresponding GFPSgs1 binding distribution. Error bars represent 95% CIs.
Figure 7.
Figure 7.. Rad52 Inhibits the Motor Activities of Sgs1
(A) Kymographs depicting GFP-Sgs1 (green) during dsDNA end resection assays on DNA pre-bound by Alexa546-Rad52 (magenta). Arrowheads highlight the position of GFP-Sgs1 at the DNA ends at the beginning of the measurement. “T” and “F” refer to the tethered and free ends of the DNA. (B) Velocity of the dsDNA resection machinery (GFP-Sgs1, Top3-Rmi1, Dna2, and RPA) in assays containing either no Rad52, 1 nM or 4 nM Alexa546-Rad52, as indicated. Error bars represent SD obtained from Gaussian fits to the data. (C) Processivity of the dsDNA resection machinery (GFP-Sgs1, Top3-Rmi1, Dna2, and RPA) in assays containing no Rad52 or 1 nM or 4 nM Alexa546-Rad52, as indicated. Error bars represent SD obtained from Gaussian fits to the data. (D) Sgs1 ATP hydrolysis activity in the presence of Rad52. Error bars represent the SD of three independent experiments. (E) Kymographs depicting the translocation activity of GFP-Sgs1 (green) on RPA-ssDNA (unlabeled) in the presence of Alexa546-Rad52 (magenta). White arrowheads in the GFP-Sgs1 image illustrate the positions at which translocating molecules of GFP-Sgs1 terminate translocation upon encountering Alexa546-Rad52. Note that the minus Rad52 data columns in (B) and (C) are reproduced from Xue et al. (2019) for comparison.
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