Manhart Carol M, Ni Xiaodan, White Martin A, Ortega Joaquin, Surtees Jennifer A, Alani Eric
Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, United States of America.
Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada.
PLoS Biol. 2017 Apr 28;15(4):e2001164. doi: 10.1371/journal.pbio.2001164. eCollection 2017 Apr.
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.
同源染色体之间的交叉互换在减数分裂前期通过全基因组范围内发生的DNA双链断裂的形成而启动。在面包酵母的主要干扰响应交叉互换途径中，这些断裂被切除以形成3'单链尾巴，这些尾巴参与同源性搜索，最终形成主要包含两条同源染色体的双Holliday连接体（dHJs）。这些dHJs通过核酸内切酶活性进行解析，以专门形成交叉互换，这对于减数分裂I中同源染色体的正确分离至关重要。最近在酵母中的遗传学、生物化学和分子研究与Mlh1-Mlh3 DNA错配修复复合物作为将dHJs解析为交叉互换的主要核酸内切酶活性的假设一致。然而，Mlh1-Mlh3核酸内切酶被激活的机制尚不清楚。在这里，我们提供证据表明，Mlh1-Mlh3的行为不像结构特异性核酸内切酶，而是形成在DNA中产生切口所需的聚合物。这一结论得到了对含有或缺乏聚合障碍的不同大小底物进行的DNA结合研究以及对核酸内切酶缺陷型和核酸内切酶 proficient型Mlh1-Mlh3的不同比例进行的核酸内切酶测定的支持。此外，Mlh1-Mlh3可以产生可重新连接的双链断裂，并形成一种活性核蛋白复合物，该复合物可以在反式中切割DNA底物。这些观察结果共同表明，Mlh1-Mlh3可能不像典型的、RuvC样的Holliday连接体解析酶那样起作用，并支持一种新模型，即Mlh1-Mlh3被加载到DNA上以形成切割DNA的活化聚合物。
