Basto Clara, Moreira-Tavares Eliana, Muhammad Ali-Akbar, Baconnais Sonia, Mazón Gerard, Le Cam Eric, Dupaigne Pauline
Genome Integrity and Cancers, UMR 9019 CNRS, Université-Paris-Saclay, Gustave Roussy, Villejuif, France.
Methods Mol Biol. 2025;2881:239-257. doi: 10.1007/978-1-0716-4280-1_12.
Homologous recombination (HR) is a high-fidelity DNA repair pathway that uses a homologous DNA sequence as a template. Recombinase proteins are the central HR players in the three kingdoms of life. RecA/RadA/Rad51 assemble on ssDNA, generated after the processing of double-strand breaks or stalled replication forks into an active and dynamic presynaptic helical nucleofilament. Presynaptic filament formation is regulated by a series of partners of the recombinase, such as scRad52/hBRCA2 mediators or anti-recombinase proteins, to form an active machinery involved in homology search, pair-matching, and invasion within homologous sequences. During homology search, but also during strand invasion, the multiprotein complexes that form the nucleofilament induce the formation of a variety of DNA intermediate states. Here we present specific approaches to study and characterize the different DNA and DNA-protein intermediates formed during homologous recombination. The combination of powerful electron microscopy and sample preparation methods provides a better understanding of these proteins' molecular activity and their interactions.
同源重组（HR）是一种高保真DNA修复途径，它使用同源DNA序列作为模板。重组酶蛋白是生命三界中同源重组的核心参与者。RecA/RadA/Rad51在双链断裂或停滞的复制叉加工后产生的单链DNA上组装成活性和动态的突触前螺旋核丝。突触前丝的形成受重组酶的一系列伙伴调节，如scRad52/hBRCA2介导物或抗重组酶蛋白，以形成参与同源序列内同源性搜索、配对匹配和侵入的活性机制。在同源性搜索期间，以及在链侵入期间，形成核丝的多蛋白复合物诱导多种DNA中间状态的形成。在这里，我们介绍了研究和表征同源重组过程中形成的不同DNA和DNA-蛋白质中间体的具体方法。强大的电子显微镜和样品制备方法的结合能更好地理解这些蛋白质的分子活性及其相互作用。
