Deveryshetty Jaigeeth, Mistry Ayush, Pangeni Sushil, Ghoneim Mohamed, Tokmina-Lukaszewska Monica, Gore Steven K, Liu Jie, Kaushik Vikas, Karunakaran Simrithaa, Taddei Angela, Heyer Wolf-Dietrich, Ha Taekjip, Bothner Brian, Antony Edwin
Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, MO, USA.
Department of Biophysics, Johns Hopkins University, Baltimore, MD, USA.
Nat Commun. 2025 Jul 21;16(1):6685. doi: 10.1038/s41467-025-61811-0.
Homologous recombination (HR) repairs double-stranded DNA breaks (DSBs) by generating single-stranded DNA (ssDNA), which is initially coated by Replication Protein A (Rpa). Rad51, a recombinase, catalyzes strand invasion but binds ssDNA with lower affinity than Rpa, necessitating mediator proteins like Rad52 (yeast) or BRCA2 (humans) for Rad51 loading. The mechanisms of this exchange remain unclear. We show that Saccharomyces cerevisiae Rad52 uses its disordered C-terminus to sort polydisperse Rad51 into discrete monomers. Using fluorescent-Rad51 and single-molecule optical tweezers, we visualize Rad52-mediated Rad51 filament formation on Rpa-coated ssDNA, preferentially at ssDNA-dsDNA junctions. Deleting the C-terminus of Rad52 disrupts Rad51 sorting and loading. Addition of the Rad51 paralog Rad55-Rad57 enhances Rad51 binding by ~60%. Despite structural differences, Rad52 and BRCA2 share conserved functional features. We propose a unified "Sort, Stack & Extend" (SSE) mechanism by which mediator proteins and paralogs coordinate Rad51 filament assembly during HR.
同源重组（HR）通过生成单链DNA（ssDNA）来修复双链DNA断裂（DSB），ssDNA最初由复制蛋白A（Rpa）包被。Rad51是一种重组酶，催化链入侵，但与Rpa相比，它与ssDNA的结合亲和力较低，因此需要像Rad52（酵母）或BRCA2（人类）这样的介导蛋白来加载Rad51。这种交换的机制尚不清楚。我们发现酿酒酵母Rad52利用其无序的C末端将多分散的Rad51分选成离散的单体。使用荧光Rad51和单分子光镊，我们观察到Rad52介导的Rad51在Rpa包被的ssDNA上形成细丝，优先在ssDNA-dsDNA交界处。删除Rad52的C末端会破坏Rad51的分选和加载。添加Rad51旁系同源物Rad55-Rad57可使Rad51的结合增强约60%。尽管结构不同，但Rad52和BRCA2具有保守的功能特征。我们提出了一种统一的“分选、堆叠和延伸”（SSE）机制，通过该机制，介导蛋白和旁系同源物在同源重组过程中协调Rad51细丝的组装。
