Putative antirecombinase Srs2 DNA helicase promotes noncrossover homologous recombination avoiding loss of heterozygosity

Abstract

DNA damage alone or DNA replication fork arrest at damaged sites may induce DNA double-strand breaks and initiate homologous recombination. This event can result in a crossover with a homologous chromosome, causing loss of heterozygosity along the chromosome. It is known that Srs2 acts as an antirecombinase at the replication fork: it is recruited by the SUMO (a small ubiquitin-related modifier)-conjugated DNA-polymerase sliding clamp (PCNA) and interferes with Rad51/Rad52-mediated homologous recombination. Here, we report that Srs2 promotes another type of homologous recombination that produces noncrossover products only, in collaboration with PCNA and Rad51. Srs2 proteins lacking the Rad51-binding domain, PCNA-SUMO-binding motifs, or ATP hydrolysis-dependent DNA helicase activity reduce this noncrossover recombination. However, the removal of either the Rad51-binding domain or the PCNA-binding motif strongly increases crossovers. Srs2 gene mutations are epistatic to mutations in the PCNA modification-related genes encoding PCNA, Siz1 (a SUMO ligase) and Rad6 (a ubiquitin-conjugating protein). Knocking out RAD51 blocked this recombination but enhanced nonhomologous end-joining. We hypothesize that, during DNA double-strand break repair, Srs2 mediates collaboration between the Rad51 nucleofilament and PCNA-SUMO and directs the heteroduplex intermediate to DNA synthesis in a moving bubble. This Rad51/Rad52/Srs2/PCNA-mediated noncrossover pathway avoids both interchromosomal crossover and imprecise end-joining, two potential paths leading to loss of heterozygosity, and contributes to genome maintenance and human health.

Keywords: NHEJ; SDSA; bubble migration.

Fig. 1.

SDSA/NHEJ assay. (A) For this assay, two different donor-alleles, 5′Δ-ura3 [with a deletion from the promoter (the 221-nt upstream of the initiation codon) to the first base of the 39th codon, residing at the ura3 locus on Chr. V] and ura3-3′Δ [the ura3 ectopic allele bearing only the region from the promoter (the 221-nt upstream of the initiation codon) to the first base of the 139th codon of the URA3 gene, integrated into the AUR1 locus on Chr. XI], were constructed (13). The plasmids with the recipient alleles, ura3-intΔnruI and ura3-intΔisceI (the internal 458-bp deletion of the URA3 gene, sealed with an NruI site and an I-SceI site, respectively, residing within the plasmid with the LEU2 marker), were introduced into the double-template strains with the ura3-3′Δ allele and 5′Δ-ura3 allele, which share 300-bp internal homology. The homologous regions between ura3-intΔ and ura3-3′Δ or 5′Δ-ura3 are 3,360 bp and 1,493 bp in length, respectively. (B) ura3-intΔisceI, ura3-intΔisceI-blunt and ura3-intΔnruI generate 4-nucleotide 3′ tails by I-SceI cleavage for pSDSA/NHEJ (with the I-SceI site) plasmid DNA, blunt ends by T4 DNA polymerase treatment following the I-SceI cleavage, and blunt ends by NruI cleavage for pSDSA/NHEJ (with the NruI site) plasmid DNA, respectively. (C) SDSA products as Ura⁺ Leu⁺ transformants bearing the URA3 plasmids (13). The 458-bp gap is repaired via SDSA. (D) NHEJ products as Ura⁻ (5FOA^R) Leu⁺ transformants bearing the ura3 plasmids (13). The retention or deletion of the I-SceI sequence was detected by PCR with the primers (arrows) (13), I-SceI nuclease treatment, and sequence determination as precise or imprecise end-joining, respectively (Table 1).

Fig. 2.

SDSA and NHEJ of homologous recombination-deficient mutants. (A) Leu⁺ transformation efficiencies with the uncut plasmid bearing ura3-intΔisceI are plotted as the transformation competencies of the cell suspensions (open circles). Ura⁺ Leu⁺ and 5-FOA^R Leu⁺ transformation efficiencies with the I-SceI–cut plasmid bearing ura3-intΔisceI are plotted as numbers of SDSA progeny (blue squares) and as numbers of NHEJ progeny (green triangles), respectively (Fig. 1). (B) The normalized frequencies (%) of SDSA events were calculated from the transformation efficiencies (Materials and Methods) and plotted (blue bars). “ND” indicates that no SDSA progeny were detected. (C) The normalized frequencies (%) of the NHEJ events were calculated from the transformation efficiencies (Materials and Methods) and plotted (green bars). rad51ΔKO lacks most of the coding region, and rad51Δw lacks the entire coding region, from the initiation codon to the last sense codon (Tables S1 and S2). rad52-KD, RD shows rad52-K117D, R148D. “NruI” and “T4 poly” indicate the use of the blunt-ended ura3-intΔnruI and ura3-intδΔisceI-blunt DNAs, respectively. The statistical analysis (Table S3): nsd, no significant difference; *P < 0.05; **P < 0.01; ***P < 0.001; two-tailed Student’s t test (vs. wild-type except for vs. rad51ΔKO); error bars = SD.

Fig. 3.

SDSA and double Holliday junction-mediated homologous recombination (dHJ-mediated HR) of various srs2 mutants. (A) The Srs2 helicase domain (blue box), including seven consensus motifs (dark blue), and the locations of the Rad51-binding domain (783-998), the N-terminal half of the Rad51-binding domain (783-859), PIP motif (1149-1156), and SIM motif (1169-1174). The 41st residue (lysine) is substituted with methionine and alanine in the srs2-K41M and srs2-K41A mutants, respectively. (B) Leu⁺ and Ura⁺ Leu⁺ (SDSA progeny) transformation efficiencies with uncut and I-SceI–cut plasmids possessing ura3-intΔisceI, respectively. (C) The normalized frequencies (%) of the SDSA events were calculated from the transformation efficiencies (Materials and Methods) and plotted (blue bars). (D) Targeted integration completed by double Holliday junction-mediated homologous recombination (Fig. S2). The plot shows the Aur^R transformation efficiencies with the uncut pRS315-Aur^R plasmid as the transformation competencies of the cell suspensions (open circles), and the Aur^R transformation efficiencies with the StuI-cut pAUR101 plasmid as the numbers of double Holliday junction-mediated homologous recombination progeny (closed triangles). (E) The normalized frequencies (%) of the double Holliday junction-mediated homologous recombination were calculated from the transformation efficiencies (Materials and Methods) and plotted (black bars). Statistical analysis (Tables S4 and S5): nsd, no significant difference; *P < 0.05; **P < 0.01; ***P < 0.001; two-tailed Student’s t test (vs. wild-type); error bars = SD.

Fig. 4.

SRS2 is epistatic to SIZ1, POL30 (PCNA), and RAD6 (PCNA modification-related genes), with respect to SDSA. (A) Leu⁺ and Ura⁺ Leu⁺ (SDSA progeny) transformation efficiencies with uncut and I-SceI–cut plasmids possessing ura3-intΔisceI, respectively. (B) The normalized frequencies (%) of the SDSA events were calculated from the transformation efficiencies (Materials and Methods) and plotted (blue bars). Statistical analysis (Table S6): nsd, no significant difference; *P < 0.05; **P < 0.01; ***P < 0.001; two-tailed Student’s t test [black letters and asterisks, vs. wild-type (except for vs. indicated genotypes); white letters and asterisks, vs. siz1 mutant]; error bars = SD.