mlh3 mutations in baker's yeast alter meiotic recombination outcomes by increasing noncrossover events genome-wide.
mlh3 mutations in baker's yeast alter meiotic recombination outcomes by increasing noncrossover events genome-wide.
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DOI：
10.1371/journal.pgen.1006974
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发表时间：
2017-08
期刊：
影响因子：
4.5
通讯作者：
Alani E
中科院分区：
文献类型：
--
作者：
Al-Sweel N;Raghavan V;Dutta A;Ajith VP;Di Vietro L;Khondakar N;Manhart CM;Surtees JA;Nishant KT;Alani E
Mlh 1-Mlh 3是一种核酸内切酶，被推测在减数分裂中起作用以将双霍利迪连接解析成交换。它也在真核DNA错配修复（MMR）中起次要作用。为了了解Mlh 1-Mlh 3在减数分裂和MMR中的功能，我们在面包酵母中分析了60个新的mlh 3等位基因。五个等位基因特异性破坏MMR，而一个（mlh 3 -32）特异性破坏减数分裂交换。纯化并表征每类的mlh 1-mlh 3代表。Mlh 1-mlh 3 -32（MMR+，交换-）和Mlh 1-mlh 3 -45（MMR-，交换+）在体外均显示野生型核酸内切酶活性。Msh 2-Msh 3是MMR中与Mlh 1-Mlh 3作用的MSH复合物，其刺激Mlh 1-mlh 3 -32的内切酶活性，但不刺激Mlh 1-mlh 3 -45，表明Mlh 1-mlh 3 -45在MSH相互作用中是缺陷的。在S288 c/YJM 789杂交背景中构建野生型和MMR+交换-、MMR-交换+、内切酶缺陷和无效mlh 3突变体的全基因组重组图谱。与野生型相比，所有的mlh 3突变体都表现出非交换事件数量的增加，这与通过替代重组途径解决重组中间体一致。我们的观察提供了一个Mlh 3的结构-功能图，揭示了蛋白质-蛋白质相互作用在调节Mlh 1-Mlh 3的酶活性中的重要性。他们还说明了如何有缺陷的减数分裂组件可以改变减数分裂重组中间体的命运，减数分裂重组途径是如何调节提供了新的见解。在减数分裂过程中，成为卵子或精子的二倍体生殖细胞经历一轮DNA复制，随后是两次连续的染色体分裂。在大多数生物中，第一次减数分裂时染色体的分离依赖于染色体同源物之间至少一次遗传交换或交叉事件。没有接受交换的同源物在第一次减数分裂时经常经历不分离，产生缺乏染色体或含有额外拷贝的配子。这些事件与人类疾病和不育有关。最近的研究表明，Mlh 1-Mlh 3复合物是一种内切核酸酶，它将重组中间体分解为交叉。有趣的是，这种复合物还在DNA错配修复（MMR）中充当媒人，以消除DNA复制错误。一个复合体如何在两个不同的过程中起作用？我们通过对面包酵母Mlh 3蛋白进行突变分析来研究这个问题。鉴定出五个突变破坏MMR但不交叉，并且一个突变破坏交叉同时维持MMR。使用生化和遗传分析的组合，以进一步表征这些突变体，我们说明蛋白质-蛋白质相互作用的Mlh 1-Mlh 3的活性的重要性。重要的是，我们的数据说明了有缺陷的减数分裂组分如何改变减数分裂重组事件的结果。它们还为不孕综合征的基础提供了新的见解。
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 wild-type, all of the mlh3 mutants showed increases in the number of noncrossover events, consistent with recombination intermediates being resolved through alternative recombination pathways. Our observations provide a structure-function map for Mlh3 that reveals the importance of protein-protein interactions in regulating Mlh1-Mlh3’s enzymatic activity. They also illustrate how defective meiotic components can alter the fate of meiotic recombination intermediates, providing new insights for how meiotic recombination pathways are regulated. During meiosis, diploid germ cells that become eggs or sperm undergo a single round of DNA replication followed by two consecutive chromosomal divisions. The segregation of chromosomes at the first meiotic division is dependent in most organisms on at least one genetic exchange, or crossover event, between chromosome homologs. Homologs that do not receive a crossover frequently undergo nondisjunction at the first meiotic division, yielding gametes that lack chromosomes or contain additional copies. Such events have been linked to human disease and infertility. Recent studies suggest that the Mlh1-Mlh3 complex is an endonuclease that resolves recombination intermediates into crossovers. Interestingly, this complex also acts as a matchmaker in DNA mismatch repair (MMR) to remove DNA replication errors. How does one complex act in two different processes? We investigated this question by performing a mutational analysis of the baker’s yeast Mlh3 protein. Five mutations were identified that disrupted MMR but not crossing over, and one mutation disrupted crossing over while maintaining MMR. Using a combination of biochemical and genetic analyses to further characterize these mutants we illustrate the importance of protein-protein interactions for Mlh1-Mlh3’s activity. Importantly, our data illustrate how defective meiotic components can alter the outcome of meiotic recombination events. They also provide new insights for the basis of infertility syndromes.
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