Genome Maintenance and Molecular Microscopy UMR8126 CNRS, Université Paris-Sud, Université Paris-Saclay, Gustave Roussy, F-94805, Villejuif Cedex, France.
Department of Microbiology and Molecular Genetics, University of California, Davis, Davis, CA, 95616-8665, USA.
Nat Commun. 2019 Sep 6;10(1):4058. doi: 10.1038/s41467-019-12082-z.
Homologous recombination (HR) uses a homologous template to accurately repair DNA double-strand breaks and stalled replication forks to maintain genome stability. During homology search, Rad51 nucleoprotein filaments probe and interact with dsDNA, forming the synaptic complex that is stabilized on a homologous sequence. Strand intertwining leads to the formation of a displacement-loop (D-loop). In yeast, Rad54 is essential for HR in vivo and required for D-loop formation in vitro, but its exact role remains to be fully elucidated. Using electron microscopy to visualize the DNA-protein complexes, here we find that Rad54 is crucial for Rad51-mediated synaptic complex formation and homology search. The Rad54-K341R ATPase-deficient mutant protein promotes formation of synaptic complexes but not D-loops and leads to the accumulation of stable heterologous associations, suggesting that the Rad54 ATPase is involved in preventing non-productive intermediates. We propose that Rad51/Rad54 form a functional unit operating in homology search, synaptic complex and D-loop formation.
同源重组（HR）利用同源模板准确修复 DNA 双链断裂和停滞的复制叉，以维持基因组稳定性。在同源性搜索过程中，Rad51 核蛋白丝与 dsDNA 探测和相互作用，形成在同源序列上稳定的联会复合物。链纠缠导致形成置换环（D-loop）。在酵母中，Rad54 对体内 HR 是必需的，并且在体外 D-loop 形成中也是必需的，但它的确切作用仍有待充分阐明。通过使用电子显微镜可视化 DNA-蛋白质复合物，我们发现 Rad54 对 Rad51 介导的联会复合物形成和同源搜索至关重要。Rad54-K341R 解旋酶缺陷突变蛋白促进联会复合物的形成，但不促进 D-loop 的形成，并导致稳定的异源复合物的积累，表明 Rad54 解旋酶参与防止非生产性中间体的形成。我们提出 Rad51/Rad54 形成一个功能单元，在同源搜索、联会复合物和 D-loop 形成中发挥作用。
