The mismatch repair and meiotic recombination endonuclease Mlh1-Mlh3 is activated by polymer formation and can cleave DNA substrates in trans

Abstract

Crossing over between homologs is initiated in meiotic prophase by the formation of DNA double-strand breaks that occur throughout the genome. In the major interference-responsive crossover pathway in baker’s yeast, these breaks are resected to form 3' single-strand tails that participate in a homology search, ultimately forming double Holliday junctions (dHJs) that primarily include both homologs. These dHJs are resolved by endonuclease activity to form exclusively crossovers, which are critical for proper homolog segregation in Meiosis I. Recent genetic, biochemical, and molecular studies in yeast are consistent with the hypothesis of Mlh1-Mlh3 DNA mismatch repair complex acting as the major endonuclease activity that resolves dHJs into crossovers. However, the mechanism by which the Mlh1-Mlh3 endonuclease is activated is unknown. Here, we provide evidence that Mlh1-Mlh3 does not behave like a structure-specific endonuclease but forms polymers required to generate nicks in DNA. This conclusion is supported by DNA binding studies performed with different-sized substrates that contain or lack polymerization barriers and endonuclease assays performed with varying ratios of endonuclease-deficient and endonuclease-proficient Mlh1-Mlh3. In addition, Mlh1-Mlh3 can generate religatable double-strand breaks and form an active nucleoprotein complex that can nick DNA substrates in trans. Together these observations argue that Mlh1-Mlh3 may not act like a canonical, RuvC-like Holliday junction resolvase and support a novel model in which Mlh1-Mlh3 is loaded onto DNA to form an activated polymer that cleaves DNA.

Cytosine deamination and base excision repair cause R-loop–induced CAG repeat fragility and instability in Saccharomyces cerevisiae

“…This mechanism could account for the partial dependence on Fcy1 and Apn1, that is, an initial nick created by processing of a deaminated cytosine could direct Mlh1-Mlh3 cleavage to the opposite strand to create a DSB. This same concerted mechanism can also occur without a preexisting nick, so another possibility is that recruitment to the DNA structure initiates MutLγ nondirectional cleavage in the vicinity (62). Our data have potentially significant implications for the Huntington's disease (HD) process because MutLγ was reported to be important in driving somatic CAG repeat instability in a HD mouse model (63).…”

“…6). Alternatively, recent results show that the MutLγ endonuclease activity can be directed by a nick to cleave the opposite strand in a concerted mechanism to create a DSB (62). This mechanism could account for the partial dependence on Fcy1 and Apn1, that is, an initial nick created by processing of a deaminated cytosine could direct Mlh1-Mlh3 cleavage to the opposite strand to create a DSB.…”

Mechanistic insight into crossing over during mouse meiosis

“…Alternatively, it has been argued that biased resolution of ligated (or unligated) substrates could be directed by proteins that promote Mlh1-Mlh3-dependent crossing over (Manhart et al, 2017). For example, the first junction formed by strand invasion and repair synthesis could accumulate proteins that stabilize this intermediate, which then direct Mlh1-Mlh3 activity in a preferred configuration, such as PCNA and Msh4-Msh5 (Manhart et al, 2017). Biochemical studies with the yeast complex suggest that it functions in a manner distinct from well-characterized structure-specific nucleases by forming higher order polymer structures (Claeys Bouuaert and Keeney, 2017;Manhart et al, 2017;Ranjha et al, 2014;Rogacheva et al, 2014), which could enforce this bias.…”

“…For example, the first junction formed by strand invasion and repair synthesis could accumulate proteins that stabilize this intermediate, which then direct Mlh1-Mlh3 activity in a preferred configuration, such as PCNA and Msh4-Msh5 (Manhart et al, 2017). Biochemical studies with the yeast complex suggest that it functions in a manner distinct from well-characterized structure-specific nucleases by forming higher order polymer structures (Claeys Bouuaert and Keeney, 2017;Manhart et al, 2017;Ranjha et al, 2014;Rogacheva et al, 2014), which could enforce this bias. Regardless of mechanism, directed resolution of double Holliday junctions appears to restrict the outcome of recombination to one particular crossover configuration, rather than the four possible outcomes (two crossover and two noncrossover) that were originally proposed (Szostak et al, 1983).…”

Genome wide analysis of meiotic recombination in yeast: For a few SNPs more

“…If the strand invasion is stabilized by the ZMM proteins (Zip1, Zip2, Zip3, Zip4, Mer3, Msh4, Msh5 and Spo16), it may be extended further by repair synthesis using the homolog and capture the second end of the DSB and form double Holliday junction 6. Biased resolution of these double Holliday junctions facilitated by the ZMM, STR (Sgs1, Top3, Rmi1), Exo1 and the Mlh1‐Mlh3 endonuclease leads to crossovers 7, 8, 9, 10, 11, 12, 13, 14. These class I crossovers show interference—a phenomena where the occurrence of a crossover event in a genetic interval makes it less likely for crossovers to occur in adjacent intervals.…”