Mlh3 mutations in baker's yeast alter meiotic recombination outcomes by increasing noncrossover events genome-wide

Mlh1-Mlh3 is an endonuclease hypothesized to act in meiosis to resolve double Holliday junctions into crossovers. It also plays a minor role in eukaryotic DNA mismatch repair (MMR). To understand how Mlh1-Mlh3 functions in both meiosis and MMR, we analyzed in baker's yeast 60 new mlh3 alleles. Five alleles specifically disrupted MMR, whereas one (mlh3-32) specifically disrupted meiotic crossing over. Mlh1-mlh3 representatives for each class were purified and characterized. Both Mlh1-mlh3-32 (MMR+, crossover-) and Mlh1-mlh3-45 (MMR-, crossover+) displayed wild-type endonuclease activities in vitro. Msh2-Msh3, an MSH complex that acts with Mlh1-Mlh3 in MMR, stimulated the endonuclease activity of Mlh1-mlh3-32 but not Mlh1-mlh3-45, suggesting that Mlh1-mlh3-45 is defective in MSH interactions. Whole genome recombination maps were constructed for wild-type and MMR+ crossover-, MMR- crossover+, endonuclease defective and null mlh3 mutants in an S288c/YJM789 hybrid background. Compared to ...Mlh1-Mlh3是一种核酸内切酶，被推测在减数分裂中发挥功能，将双重霍利迪联结体（double Holliday junctions）解析为交换产物。该复合物在真核生物DNA错配修复（DNA mismatch repair, MMR）中亦发挥次要作用。为阐明Mlh1-Mlh3在减数分裂与错配修复中的作用机制，我们以酿酒酵母（baker's yeast）为模型，分析了60个全新的mlh3等位基因。其中5个等位基因可特异性破坏错配修复功能，而1个等位基因（mlh3-32）可特异性阻断减数分裂交换过程。我们针对每一类等位基因对应的Mlh1-Mlh3复合物进行了纯化与功能表征。体外实验显示，Mlh1-Mlh3-32（错配修复功能正常、减数分裂交换缺陷型）与Mlh1-Mlh3-45（错配修复缺陷型、减数分裂交换功能正常）均表现出野生型水平的核酸内切酶活性。作为在错配修复中与Mlh1-Mlh3协同发挥功能的MSH复合物（MSH complex），Msh2-Msh3可增强Mlh1-Mlh3-32的核酸内切酶活性，但对Mlh1-Mlh3-45无此促进作用，这提示Mlh1-Mlh3-45存在与MSH复合物相互作用的缺陷。我们在S288c/YJM789杂交背景下，为野生型菌株以及错配修复正常/交换缺陷型、错配修复缺陷/交换正常型、核酸内切酶缺陷型以及敲除型mlh3突变菌株构建了全基因组重组图谱。相较于……