Central to homologous recombination in eukaryotes is the RAD51 recombinase, which forms helical nucleoprotein filaments on single-stranded DNA (ssDNA) and catalyzes strand invasion with homologous duplex DNA. Various regulatory proteins assist this reaction including the RAD51 paralogs. We recently discovered that a RAD51 paralog complex from C. elegans, RFS-1/RIP-1, functions predominantly downstream of filament assembly by binding and remodeling RAD-51-ssDNA filaments to a conformation more proficient for strand exchange. Here, we demonstrate that RFS-1/RIP-1 acts by shutting down RAD-51 dissociation from ssDNA. Using stopped-flow experiments, we show that RFS-1/RIP-1 confers this dramatic stabilization by capping the 5′ end of RAD-51-ssDNA filaments. Filament end capping propagates a stabilizing effect with a 5′→3′ polarity approximately 40 nucleotides along individual filaments. Finally, we discover that filament capping and stabilization are dependent on nucleotide binding, but not hydrolysis by RFS-1/RIP-1. These data define the mechanism of RAD51 filament remodeling by RAD51 paralogs.

真核生物同源重组的核心是 RAD51 重组酶，它在单链 DNA (ssDNA) 上形成螺旋核蛋白丝，并催化同源双链 DNA 的链入侵。多种调节蛋白有助于该反应，包括 RAD51 旁系同源物。我们最近发现，来自秀丽隐杆线虫的 RAD51 旁系同源复合物 RFS-1/RIP-1 通过将 RAD-51-ssDNA 丝结合和重塑为更适合链交换的构象，主要在丝组装下游发挥作用。在这里，我们证明 RFS-1/RIP-1 通过关闭 RAD-51 与 ssDNA 的解离来发挥作用。通过停流实验，我们表明 RFS-1/RIP-1 通过封盖 RAD-51-ssDNA 丝的 5' 端来赋予这种显着的稳定性。细丝封端以 5'→3' 极性沿着单个细丝传播大约 40 个核苷酸的稳定效应。最后，我们发现丝的加帽和稳定依赖于核苷酸结合，而不是 RFS-1/RIP-1 的水解。这些数据定义了 RAD51 旁系同源物对 RAD51 丝进行重塑的机制。