Institute for Research in Biomedicine, Faculty of Biomedical Sciences, Università della Svizzera italiana (USI), Bellinzona, Switzerland.
Department of Biology, Institute of Biochemistry, Eidgenössische Technische Hochschule (ETH), Zürich, Switzerland.
Nature. 2020 Oct;586(7830):618-622. doi: 10.1038/s41586-020-2592-2. Epub 2020 Aug 19.
During prophase of the first meiotic division, cells deliberately break their DNA. These DNA breaks are repaired by homologous recombination, which facilitates proper chromosome segregation and enables the reciprocal exchange of DNA segments between homologous chromosomes. A pathway that depends on the MLH1-MLH3 (MutLγ) nuclease has been implicated in the biased processing of meiotic recombination intermediates into crossovers by an unknown mechanism. Here we have biochemically reconstituted key elements of this pro-crossover pathway. We show that human MSH4-MSH5 (MutSγ), which supports crossing over, binds branched recombination intermediates and associates with MutLγ, stabilizing the ensemble at joint molecule structures and adjacent double-stranded DNA. MutSγ directly stimulates DNA cleavage by the MutLγ endonuclease. MutLγ activity is further stimulated by EXO1, but only when MutSγ is present. Replication factor C (RFC) and the proliferating cell nuclear antigen (PCNA) are additional components of the nuclease ensemble, thereby triggering crossing-over. Saccharomyces cerevisiae strains in which MutLγ cannot interact with PCNA present defects in forming crossovers. Finally, the MutLγ-MutSγ-EXO1-RFC-PCNA nuclease ensemble preferentially cleaves DNA with Holliday junctions, but shows no canonical resolvase activity. Instead, it probably processes meiotic recombination intermediates by nicking double-stranded DNA adjacent to the junction points. As DNA nicking by MutLγ depends on its co-factors, the asymmetric distribution of MutSγ and RFC-PCNA on meiotic recombination intermediates may drive biased DNA cleavage. This mode of MutLγ nuclease activation might explain crossover-specific processing of Holliday junctions or their precursors in meiotic chromosomes.
在第一次减数分裂前期，细胞故意破坏其 DNA。这些 DNA 断裂由同源重组修复，促进染色体正确分离，并允许同源染色体之间的 DNA 片段发生交互交换。一种依赖于 MLH1-MLH3（MutLγ）核酸内切酶的途径，通过未知的机制，有利于偏向地将减数分裂重组中间体加工成交叉。在这里，我们通过生化方法重建了这个前交叉途径的关键要素。我们表明，支持交叉的人 MSH4-MSH5（MutSγ）与分支重组中间体结合，并与 MutLγ 相关联，将联合体稳定在联合分子结构和相邻的双链 DNA 上。MutSγ 直接刺激 MutLγ 内切核酸酶的 DNA 切割。MutLγ 活性进一步受到 EXO1 的刺激，但只有在 MutSγ 存在的情况下才会受到刺激。复制因子 C（RFC）和增殖细胞核抗原（PCNA）是核酸内切酶联合体的其他组成部分，从而触发交叉。无法与 PCNA 相互作用的酿酒酵母菌株在形成交叉方面存在缺陷。最后，MutLγ-MutSγ-EXO1-RFC-PCNA 核酸内切酶联合体优先切割带有 Holliday 连接点的 DNA，但没有典型的解旋酶活性。相反，它可能通过在连接点附近切割双链 DNA 来处理减数分裂重组中间体。由于 MutLγ 的 DNA 切口依赖于其辅助因子，因此 MutSγ 和 RFC-PCNA 在减数分裂重组中间体上的不对称分布可能会驱动偏向的 DNA 切口。这种 MutLγ 核酸内切酶激活模式可能解释了减数分裂染色体中 Holliday 连接点或其前体的交叉特异性处理。
