Tolerance of DNA Mismatches in Dmc1 Recombinase-mediated DNA Strand Exchange.

Recombination between homologous chromosomes is required for the faithful meiotic segregation of chromosomes and leads to the generation of genetic diversity. The conserved meiosis-specific Dmc1 recombinase catalyzes homologous recombination triggered by DNA double strand breaks through the exchange of parental DNA sequences. Although providing an efficient rate of DNA strand exchange between polymorphic alleles, Dmc1 must also guard against recombination between divergent sequences. How DNA mismatches affect Dmc1-mediated DNA strand exchange is not understood. We have used fluorescence resonance energy transfer to study the mechanism of Dmc1-mediated strand exchange between DNA oligonucleotides with different degrees of heterology. The efficiency of strand exchange is highly sensitive to the location, type, and distribution of mismatches. Mismatches near the 3' end of the initiating DNA strand have a small effect, whereas most mismatches near the 5' end impede strand exchange dramatically. The Hop2-Mnd1 protein complex stimulates Dmc1-catalyzed strand exchange on homologous DNA or containing a single mismatch. We observed that Dmc1 can reject divergent DNA sequences while bypassing a few mismatches in the DNA sequence. Our findings have important implications in understanding meiotic recombination. First, Dmc1 acts as an initial barrier for heterologous recombination, with the mismatch repair system providing a second level of proofreading, to ensure that ectopic sequences are not recombined. Second, Dmc1 stepping over infrequent mismatches is likely critical for allowing recombination between the polymorphic sequences of homologous chromosomes, thus contributing to gene conversion and genetic diversity.

Functional analysis of the interaction between the mismatch repair protein MutS and the replication processivity factor ß clamp in Pseudomonas aeruginosa.

Interaction between MutS and the replication factor ß clamp has been extensively studied in a Mismatch Repair context; however, its functional consequences are not well understood. We have analyzed the role of the MutS-ß clamp interaction in Pseudomonas aeruginosa by characterizing a ß clamp binding motif mutant, denominated MutSß, which does not interact with the replication factor. A detailed characterization of P. aeruginosa strain PAO1 harboring a chromosomal mutSß allele demonstrated that this mutant strain exhibited mutation rates to rifampicin and ciprofloxacin resistance comparable to that of the parental strain. mutSß PAO1 was as proficient as the parental strain for DNA repair under highly mutagenic conditions imposed by the adenine base analog 2-aminopurine. In addition, using a tetracycline resistance reversion assay to assess the repair of a frameshift mutation, we determined that the parental and mutSß strains exhibited similar reversion rates. Our results clearly indicate that the MutS-ß clamp interaction does not have a central role in the methylation-independent Mismatch Repair of P. aeruginosa.