RPA Stabilization of Single-Stranded DNA Is Critical for Break-Induced Replication

Abstract

DNA double-strand breaks (DSBs) are cytotoxic lesions that must be accurately repaired to maintain genome stability. Replication protein A (RPA) plays an important role in homology-dependent repair of DSBs by protecting the single-stranded DNA (ssDNA) intermediates formed by end resection and by facilitating Rad51 loading. We found that hypomorphic mutants of RFA1 that support intra-chromosomal homologous recombination are profoundly defective for repair processes involving long tracts of DNA synthesis, in particular break-induced replication (BIR). The BIR defects of the rfa1 mutants could be partially suppressed by eliminating the Sgs1-Dna2 resection pathway, suggesting that Dna2 nuclease attacks the ssDNA formed during end resection when not fully protected by RPA. Overexpression of Rad51 was also found to suppress the rfa1 BIR defects. We suggest that Rad51 binding to the ssDNA formed by excessive end resection and during D-loop migration can partially compensate for dysfunctional RPA.

Keywords: DSB repair; Dna2; RPA; Rad51; break-induced replication; homologous recombination.

Figure 1. The rfa1 mutants are proficient for intra-chromosomal HR

A. Schematic of the MAT locus showing the location of the HO cut site and the StyI restriction sites used to distinguish between MATa and MATα alleles. The red line indicates the location of the probe used. B. Genomic blot of DNA extracted from the indicated strains at different times after HO induction. C. Cartoon of the direct repeat recombination reporter. After I-SceI cleavage repair can occur by gene conversion (GC), retaining the TRP1 marker between the repeats, or by SSA resulting in deletion of TRP1 and one of the repeats. Repair products are mostly Ade⁺. D. The frequency of GC and SSA repair of the indicated strains is shown. WT refers to wild type. Error bars indicate SD (n=4–6). * indicates p values of <0.05. See also Fig S1

Figure 2. BIR is defective in rfa1 hypomorphic mutants

A. Schematic of the ectopic BIR assay. The locations of the primers used to monitor DSB formation (D1, D2), BIR product (P1, P2) and control primers (C1, C2) are indicated by horizontal arrows. B. BIR frequencies of the indicated strains with the donor cassette located 15, 70 or 128 kb from the telomere. Error bars show SD (n=3–5). C. Detection of BIR intermediates by PCR. Error bars show SD (n=3). D. Schematic showing the predicted sizes of genomic EcoRV fragments before and after BIR. E. Genomic blot of the indicated strains showing formation of completed BIR product in real time. See also Figs S2 and S3

Figure 3. The rfa1-S373P mutant is defective for long-tract gene conversion

A. Schematic showing the recipient LEU2 gene targeted by HO on Chr V (black line) and the donor cassettes on Chr III (grey lines) with varying distance between LE and U2. B. Percent survival of the indicated strains. Error bars show the SD (n=3).

Figure 4. Rad51 over-expression, but not mph1Δ, suppresses the rfa1 BIR defect

A. Percent BIR for the indicated strains expressing empty vector (EV) or RAD51 from a high copy number vector. Error bars show the SD (n=3). B. Rad51 enrichment at sequence 2 kb upstream of the DSB in the indicated strains, 0, 2 or 4 h after HO induction. Error bars show SD (n=3). C. Percent repair for the indicated 5 kb gap strains expressing EV or RAD51 from a high copy number vector. Error bars show SD (n=3). D. Percent BIR for the indicated MPH1 and mph1Δ strains. Error bars show SD (n=3). See also Figs S4 and S5

Figure 5. 3′ strand loss contributes to the BIR defects of the rfa1 mutants

A. Fraction of recipient locus retained in the indicated strains at 2, 6 and 10 h after HO induction. B. Percent BIR for the indicated strains, error bars show SD (n=3). * Indicates a p value of <0.05; ** indicates a p value of <0.01.

Figure 6. Model for Break Repair and BIR

Break repair involves short ssDNA intermediates that are less susceptible to degradation when RPA is dysfunctional. By contrast, BIR involves long-lived ssDNA intermediates formed by end resection and migrating D-loop synthesis that are more dependent on fully functional RPA for protection from degradation. Naked ssDNA is prone to base damage or hydrolysis and forms secondary structure that could be targeted by structure-selective nucleases thereby destroying recombination intermediates. Rad51 OE suppresses the BIR defect of the rfa1 mutants, pre or post-synaptically, by protecting ssDNA and promoting strand invasion.