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Homologous recombination is a DNA damage repair and tolerance pathway that prevents genomic instability and is conserved from E. coli to H. sapiens. Multiple steps within the pathway are subject to regulation and can lead to a variety of subpathways and outcomes. A central step to homologous recombination is the formation of the Rad51 filament on single-stranded DNA flanking the initiating lesion. Rad51 utilizes the DNA sequence information to perform its major functions: homology search, invasion of duplex DNA, and DNA strand exchange. In S. cerevisiae Rad51 filaments are assembled or stabilized with the help of mediators and positive regulators (Rad52, Rad55-57, Shu Complex, Mei5-Sae3). In opposition, assembly of Rad51 filaments is impeded by RPA and filaments are actively disassembled by the helicase Srs2. How mediators like Rad55-57 function to stabilize Rad51 filaments remains unknown. Previous work showed that Rad55 and Rad57 share sequence homology with Rad51 in regions important for nucleotide cofactor interaction, and that they form a heterodimer that mitigates the disassembly of Rad51 filaments by Srs2. To understand the mechanism that underlies how the Rad55-57 dimer stabilizes Rad51 filaments, several complementary approaches were used. Mutational changes to the conserved nucleotide cofactor binding sites explored their importance to the biological function of Rad55-57. Unlike Rad51, ATP-hydrolysis is largely dispensable for Rad55-57, but ATP-binding is essential although not in all situations. Biochemical study of Rad55-57 revealed that nucleotide cofactor can affect its interaction with Rad51 and that Rad51 ATPase activity on single-stranded DNA is inhibited. The Rad51 filament is a dynamic multi-protein structure influenced by its components and other interacting proteins. Pulldown experiments with the Rad55-57 heterodimer show that more Rad57 binds within the Rad51 filament than Rad55. Lastly, to determine the kinetics of Rad51 filament disassembly by Srs2, measurement of single-molecule DNA length changes of Rad51 filaments were observed in real-time by magnetic tweezers. The work summarized here suggests that Rad55 and Rad57 are regulated by their interaction with nucleotide cofactor and contribute a complex role in the Rad51 filament.
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