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Although Rad51 is the key protein in homologous recombination (HR), a major DNA double-strand break repair pathway, several auxiliary factors interact with Rad51 to promote productive HR. We present an interdisciplinary characterization of the interaction between Rad51 and Swi5-Sfr1, a conserved auxiliary factor. Two distinct sites within the intrinsically disordered N-terminus of Sfr1 (Sfr1N) were found to cooperatively bind Rad51. Deletion of this domain impaired Rad51 stimulation in vitro and rendered cells sensitive to DNA damage. By contrast, amino acid-substitution mutants, which had comparable biochemical defects, could promote DNA repair, suggesting that Sfr1N has another role in addition to Rad51 binding. Unexpectedly, the DNA repair observed in these mutants was dependent on Rad55-Rad57, another auxiliary factor complex hitherto thought to function independently of Swi5-Sfr1. When combined with the finding that they form a higher-order complex, our results imply that Swi5-Sfr1 and Rad55-Rad57 can collaboratively stimulate Rad51 in Schizosaccharomyces pombe.

Abstract: Misregulation of DNA repair is associated with genetic instability and tumorigenesis. To preserve the integrity of the genome, eukaryotic cells have evolved extremely intricate mechanisms for repairing DNA damage. One type of DNA lesion is a double-strand break (DSB), which is highly toxic when unrepaired. Repair of DSBs can occur through multiple mechanisms. Aside from religating the DNA ends, a homologous template can be used for repair in a process called homologous recombination (HR). One key step in committing to HR is the formation of Rad51 filaments, which perform the homology search and strand invasion steps. In S. cerevisiae, Srs2 is a key regulator of Rad51 filament formation and disassembly. In this review, we highlight potential candidates of Srs2 orthologues in human cells, and we discuss recent advances in understanding how Srs2''s so-called “anti-recombinase” activity is regulated. [Copyright &y& Elsevier]

Homologous recombination (HR) is a mechanism conserved from bacteria to humans essential for the accurate repair of DNA double-stranded breaks, and maintenance of genome integrity. In eukaryotes, the key DNA transactions in HR are catalyzed by the Rad51 recombinase, assisted by a host of regulatory factors including mediators such as Rad52 and Rad51 paralogs. Rad51 paralogs play a crucial role in regulating proper levels of HR, and mutations in the human counterparts have been associated with diseases such as cancer and Fanconi Anemia. In this review, we focus on the Saccharomyces cerevisiae Rad51 paralog complex Rad55–Rad57, which has served as a model for understanding the conserved role of Rad51 paralogs in higher eukaryotes. Here, we discuss the results from early genetic studies, biochemical assays, and new single-molecule observations that have together contributed to our current understanding of the molecular role of Rad55–Rad57 in HR. [ABSTRACT FROM AUTHOR]

Homologous recombination (HR) is essential for maintenance of genome integrity. Rad51 paralogs fulfill a conserved but undefined role in HR, and their mutations are associated with increased cancer risk in humans. Here, we use single-molecule imaging to reveal that the Saccharomyces cerevisiae Rad51 paralog complex Rad55-Rad57 promotes assembly of Rad51 recombinase filament through transient interactions, providing evidence that it acts like a classical molecular chaperone. Srs2 is an ATP-dependent anti-recombinase that downregulates HR by actively dismantling Rad51 filaments. Contrary to the current model, we find that Rad55-Rad57 does not physically block the movement of Srs2. Instead, Rad55-Rad57 promotes rapid re-assembly of Rad51 filaments after their disruption by Srs2. Our findings support a model in which Rad51 is in flux between free and single-stranded DNA (ssDNA)-bound states, the rate of which is controlled dynamically though the opposing actions of Rad55-Rad57 and Srs2., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Homologous recombination (HR) is a conserved mechanism essential for the accurate repair of DNA double stranded breaks and the exchange of genetic information during meiosis. The key steps in HR are carried out by the RecA/Rad51 class of recombinases, which form a helical filament on single-stranded DNA (ssDNA) and catalyze homology search and strand exchange with a complementary duplex DNA target. In eukaryotes, assembly of the Rad51-ssDNA filament requires regulatory factors called mediators, including Rad51 paralogs. A mechanistic understanding of the role of Rad51 paralogs in HR has been hampered by the transient and diverse nature of intermediates formed with the Rad51-ssDNA filament, which cannot be resolved by traditional ensemble methods. The biochemical characterization of Rad51 paralogs, including the S. cerevisiae complex Rad55-Rad57 has also been limited by their propensity to aggregate. Here we describe the preparation of monodisperse GFP-tagged Rad55-Rad57 complex and the methodology for its analysis in our single-molecule DNA curtain assay., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Although Rad51 is the key protein in homologous recombination (HR), a major DNA double-strand break repair pathway, several auxiliary factors interact with Rad51 to promote productive HR. We present an interdisciplinary characterization of the interaction between Rad51 and Swi5-Sfr1, a conserved auxiliary factor. Two distinct sites within the intrinsically disordered N-terminus of Sfr1 (Sfr1N) were found to cooperatively bind Rad51. Deletion of this domain impaired Rad51 stimulation in vitro and rendered cells sensitive to DNA damage. By contrast, amino acid-substitution mutants, which had comparable biochemical defects, could promote DNA repair, suggesting that Sfr1N has another role in addition to Rad51 binding. Unexpectedly, the DNA repair observed in these mutants was dependent on Rad55-Rad57, another auxiliary factor complex hitherto thought to function independently of Swi5-Sfr1. When combined with the finding that they form a higher-order complex, our results imply that Swi5-Sfr1 and Rad55-Rad57 can collaboratively stimulate Rad51 in Schizosaccharomyces pombe ., Competing Interests: BA, MS, NA, MK, KI, TM, SK, YM, HT, MT, HT, HI No competing interests declared, (© 2020, Argunhan et al.)