Mph1 and Mus81-Mms4 prevent aberrant processing of mitotic recombination intermediates

Abstract

Homology-dependent repair of double-strand breaks (DSBs) from nonsister templates has the potential to generate loss of heterozygosity or genome rearrangements. Here we show that the Saccharomyces cerevisiae Mph1 helicase prevents crossovers between ectopic sequences by removing substrates for Mus81-Mms4 or Rad1-Rad10 cleavage. A role for Yen1 is only apparent in the absence of Mus81. Cells lacking Mph1 and the three nucleases are highly defective in the repair of a single DSB, suggesting that the recombination intermediates that accumulate cannot be processed by the Sgs1-Top3-Rmi1 complex (STR). Consistent with this hypothesis, ectopic joint molecules (JMs) accumulate transiently in the mph1Δ mutant and persistently when Mus81 is eliminated. Furthermore, the ectopic JMs formed in the mus81Δ mutant contain a single Holliday junction (HJ) explaining why STR is unable to process them. We suggest that Mph1 and Mus81-Mms4 recognize an early strand exchange intermediate and direct repair to noncrossover or crossover outcomes, respectively.

Figure 1. Mph1 prevents CO between ectopic repeats

A. Schematic of the assay used to detect recombinants formed between ectopic ura3 repeats. CO products are detected by the formation of novel ApaLI (A)/PvuII (P) restriction fragments of 10.7 and 8.6 kb. B. The efficiency of HO cleavage and repair was determined by PCR amplification of the donor and recipient ura3 loci followed by BamHI digestion. C. Southern blot analysis of DNA extracted from cells of the indicated genotype following HO induction. D. The mean CO values at 8 and 24 h are shown with the 8 h value normalized to the repair efficiency and the 24 h value normalized to PE. PE was determined from 4-11 trials and COs were quantified from 3-8 trials. E. Recombinant distribution for the indicated strains normalized to the PE. Error bars show standard error of the mean (SEM) for both PE and CO. See also Figures S1 and S2.

Figure 2. Mph1, Mus81-Mms4, Rad1-Rad10 and Yen1 are required for DSB repair

A. For each strain the fraction of CO products assessed 24 h after HO induction by Southern blot hybridization was normalized to the PE (see Figure 1). Mean CO values were determined from 2-8 trials and PE from 3-11 trials of each strain, and error bars show SEM. B. Physical analysis to determine the percent crossovers among survivors (left graph) and normalized to the PE (right graph). Mean PE was determined from 3-11 trials and error bars show SEM. See also Figure S3.

Figure 3. Joint molecules accumulate in mph1Δ, mus81Δ and sgs1Δ mutants

A. Schematic showing the expected sizes of parental fragments and joint molecules after digestion with ApaLI and PvuII, and their expected migration by neutral 2D gel electrophoresis. B. 2D gel analysis of DNA isolated from the indicated strains 1.5, 2.5 or 4.5 h after HO induction. The orange and green triangles correspond to inter-sister and ectopic JM, respectively; * corresponds to HO collapsed inter-sister JMs. C. An IS-JM forms by invasion of an uncut sister chromatid by the HO cut sister. If the IS-JM is not fully gap filled and ligated then HO would cleave only the newly synthesized strand and its complement and not the D-loop. Branch migration of the junction towards the DSB would release the junction resulting in Y-shaped molecules of 11.5 or 9.5 kb. Ch-specific probes were used to verify formation of inter-sister JMs.

Figure 4. Ectopic JMs recovered from the mus81Δ mutant are connected by a single HJ

A. Native-native and native-denaturing 2D gel electrophoresis of genomic DNA isolated from the mus81Δ mutant. The ectopic JM resolves as four spots corresponding to parental and recombinant strands in the denaturing gel. B. Cartoon representation of the 2D gels. C. Equal representation of parental and recombinant strands is indicative of a sHJ, whereas dHJs are denatured to parental only strands by alkaline gel electrophoresis. D. The Southern blot membrane was sequentially probed with probes specific to Ch II or Ch V that hybridize to only one of the two parental spots and one of the two recombinant spots and to the URA3 probe that hybridizes with all parental and recombinant strands.

Figure 5. Mph1 prevents inter-homolog recombination by removing substrates for Mus81-Mms4

A. Schematic of chromosome (Ch.) XV showing the I-SceI cut site insertion creating the ade2-I allele, the asterisk notes the location of the ade2-n mutation present on the other homolog. Hph and Nat cassettes replace the HIS3 ORF 150 kb downstream of the ade2 locus. B. Distribution of NCO, CO and BIR products for red/white-sectored colonies normalized to the PE for each of the indicated strains. PE was determined from 4 trials and error bars show SEM. See also Figures S4-6.

Figure 6. Model showing the predicted substrates for Mph1, Mus81-Mms4, Rad1-Rad10 and Yen1 during ectopic recombination

NCOs result from D-loop disassembly by Mph1 or dHJ dissolution by STR. Mus81-Mms4 cuts the D-loop intermediate and captured cleaved D-loop to yield COs. Rad1-Rad10 cuts at the heterology boundary of the captured D-loop before or after cleavage by Mus81-Mms4 to generate COs. If Mus81-Mms4 fails to cut, the intact HJ can be cut by Yen1 to generate NCO or CO products. In the absence of Mph1 and Mus81, the stable D-loop intermediate can undergo extensive DNA synthesis (BIR); without Mus81 and Yen1, sHJ intermediates persist.