Mph1 requires mismatch repair-independent and -dependent functions of MutSalpha to regulate crossover formation during homologous recombination repair

Abstract

In budding yeast the DNA helicase Mph1 prevents genome rearrangements during ectopic homologous recombination (HR) by suppressing the formation of crossovers (COs). Here we show that during ectopic HR repair, the anti-CO function of Mph1 is intricately associated with the mismatch repair (MMR) factor, MutSalpha. In particular, during HR repair using a completely homologous substrate, we reveal an MMR-independent function of MutSalpha in generating COs that is specifically antagonized by Mph1, but not Sgs1. In contrast, both Mph1 and MutSalpha are required to efficiently suppress COs in the presence of a homeologous substrate. Mph1 acts redundantly with Sgs1 in this respect since mph1Delta sgs1Delta double mutant cells pheno-copy MutSalpha mutants and completely fail to discriminate homologous and homeologous sequences during HR repair. However, this defect of mph1Delta sgs1Delta cells is not due to an inability to carry out MMR but rather is accompanied by elevated levels of gene conversion (GC) and bi-directional GC tracts specifically in non-crossover products. Models describing how Mph1, MutSalpha and Sgs1 act in concert to suppress genome rearrangements during ectopic HR repair are discussed.

Figure 1.

Schematic diagram showing the outcomes of different homologous recombination repair pathways and the proposed steps in which Mph1, MutSα and Sgs1 act. During homeologous recombination repair, MutSα specifically suppresses the formation of COs by inhibiting Double HJ formation (yellow box). However, during homologous recombination, MutSα-dependent COs are generated that do not require the MMR functions of MutSα and are suppressed by the actions of Mph1 (green box). Tracts of DNA synthesis are shown by dotted blue lines with arrowheads. The resolution of HJs in one of two orientations is shown by magenta arrowheads. See text for details.

Figure 2.

(A) Schematic diagram showing plasmid-break repair assay in which pADE2(400/400) is repaired using endogenous ADE2 locus. HR repair of pADE2(400/400) is mediated via a 800-bp fragment comprising residues 200–999 of the ADE2 open reading frame. pADE2(400/400) is linearized at a unique Hpa1 site which bisects the ADE2 fragment into two 400 bp regions of homology to ADE2. The structures of crossover and non-crossover repair products are shown. (B) Confirmation of repair products. Left panel: Integration of pADE2(400/400) into the ADE2 locus in CO events was confirmed by Southern analysis; dotted line labeled p in (A) indicates the sequence used as a probe and the sizes in parentheses indicate the predicted BamHI fragments detected in wild-type (lane 2) and six independent CO products (lanes 3–8). Shown also is genomic DNA from ade2Δ cells (lane 1). Right panels: Intact circular pADE2(400/400) plasmid was recovered from NCO products and analyzed by BamHI or SnaBI and HpaI digestion, as indicated. Predicted sizes of restriction fragments are shown on the right of each panel. (C) Genetic requirements for the repair of HpaI-linearized pADE2(400/400). See text for details.

Figure 3.

The anti-CO function of Mph1, but not Sgs1, requires Msh2. (A and B) CO frequency during repair of pADE2(400/400) in various genetic backgrounds, as indicated. Bars are means with standard deviations.

Figure 4.

The anti-CO function of Mph1 is dependent on MutSα but independent of MutSβ. (A) CO frequency during repair of pADE2(400/400) in various genetic backgrounds, as indicated. (B) CO frequency during repair of pADE2(400/400) in the presence of various msh2 alleles in the presence or absence of Mph1. (C) CO frequency during repair of pADE2(400/400) in the presence of various msh6 alleles in the presence or absence of Mph1. Bars are means with standard deviations. See text for details.

Figure 5.

Homeology-mediated suppression of COs is defective in mph1Δ sgsΔ cells. (A) Left panel: Schematic diagram showing the derivation of pADE2(1 bp/mis) in which the ADE2 targeting fragment of pADE2(400/400) has been replaced with a modified version containing 10 single base substitutions as indicated by vertical black bars. The unique HpaI site present in pADE2(400/400) is present in pADE2(1 bp/mis). Right panel: Comparison of CO frequencies arising from the repair of pADE2(400/400) versus pADE2(1 bp/mis) in various genetic backgrounds, as indicated. (B) Upper panel: Comparison of CO frequencies arising from the repair of pADE2(400/400) versus pADE2(1 bp/mis) in various genetic backgrounds, as indicated. Lower panel: Fold-change in mean CO frequencies from upper panel comparing homologous [pADE2(400/400)] and homeologous [pADE2(1bp/mis)] repair substrates. Level indicative of no-change (1-fold) is shown by a dotted line. (C) Absolute CO and NCO repair efficiencies following correction for transformation efficiency for repair of either pADE2(400/400) or pADE2(1 bp/mis) in different genetic backgrounds, as indicated. * indicates those datasets that share common P–values.

Figure 6.

mph1Δ sgs1Δ cells do not have an overt defect in mismatch recognition but display altered processing of non-crossover products. (A) Upper panel: Schematic diagram showing the formation of CO products resulting from the repair of pADE2(1 bp/mis). The positions of single bases (labeled 0–9) differing from the wild-type ADE2 sequence are indicated by vertical black lines. The location of the HpaI-induced break is indicated by an open arrowhead. Black arrows indicate primers used to amplify by PCR the indicated fragments from CO products for marker analysis. Lower panel: Status of each of the markers 0–9 in individual repair products from different genetic backgrounds, as indicated. Number of individual repair products analyzed from each genetic background is shown in parentheses. (B) Upper panel: Schematic diagram showing the formation of NCO products resulting from the repair of pADE2(1 bp/mis). The position of single bases (labeled 0–9) differing from the wild-type ADE2 sequence are indicated by vertical black lines. The location of the HpaI-induced break is indicated by an open arrowhead. Black arrows indicate primers used to amplify by PCR the indicated fragment from NCO products for marker analysis. Lower panel: Status of each of the markers 0–9 in individual repair products from different genetic backgrounds, as indicated. Number of individual repair products analyzed from each genetic background is shown in parentheses. To aid comparison between strains in (A) and (B), the data for each strain has been proportionally scaled in order that the total number of repair products occupy the same area.

Figure 7.

Non-crossover products from mph1Δ sgs1Δ cells have elevated frequencies of gene conversion and bi-directional gene conversion tracts. (A) Upper panel: Gene conversion frequencies for individual markers 0–9 in CO products resulting from the repair of pADE2(1 bp/mis) in various genetic backgrounds, as determined from Figure 6A. Lower panel: Gene conversion frequencies for individual markers 0–9 in NCO products resulting from the repair of pADE2(1 bp/mis) in various genetic backgrounds, as determined from Figure 6B. (B) Upper panel: Gene conversion tract directionality of CO products resulting from the repair of pADE2(1 bp/mis) in various genetic backgrounds, as determined from Figure 6A. Lower panel: Gene conversion tract directionality of NCO products resulting from the repair of pADE2(1 bp/mis) in various genetic backgrounds, as determined from Figure 6B.