Nucleotide proofreading functions by nematode RAD51 paralogs facilitate optimal RAD51 filament function

The RAD51 recombinase assembles as helical nucleoprotein filaments on single-stranded DNA (ssDNA) and mediates invasion and strand exchange with homologous duplex DNA (dsDNA) during homologous recombination (HR), as well as protection and restart of stalled replication forks. Strand invasion by RAD51-ssDNA complexes depends on ATP binding. However, RAD51 can bind ssDNA in non-productive ADP-bound or nucleotide-free states, and ATP-RAD51-ssDNA complexes hydrolyse ATP over time. Here, we define unappreciated mechanisms by which the RAD51 paralog complex RFS-1/RIP-1 limits the accumulation of RAD-51-ssDNA complexes with unfavorable nucleotide content. We find RAD51 paralogs promote the turnover of ADP-bound RAD-51 from ssDNA, in striking contrast to their ability to stabilize productive ATP-bound RAD-51 nucleoprotein filaments. In addition, RFS-1/RIP-1 inhibits binding of nucleotide-free RAD-51 to ssDNA. We propose that ‘nucleotide proofreading’ activities of RAD51 paralogs co-operate to ensure the enrichment of active, ATP-bound RAD-51 filaments on ssDNA to promote HR.

The Role of the Rad55–Rad57 Complex in DNA Repair

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.

The homologous recombination complex of the Rad51 paralogs Rad55-Rad57 avoids translesion DNA polymerase recruitment and counterbalances mutagenesis induced by UV radiation

Bypass of DNA lesions that block replicative polymerases during DNA replication relies on several DNA damage tolerance pathways. The error-prone translesion synthesis (TLS) pathway involves specialized DNA polymerases that incorporate nucleotides in front of base lesions. The template switching and the homologous recombination (HR) pathways are mostly error-free because the bypass is performed by using typically the sister chromatid as a template. This is promoted by the Rad51 recombinase that forms nucleoprotein filaments on single-strand DNA (ssDNA). The balance between error-prone and error-free pathways controls the level of mutagenesis. In yeast, the Rad55-Rad57 complex of Rad51 paralogs is required for Rad51 filament formation and stability, notably by counteracting the Srs2 antirecombinase. Several reports showed that Rad55-Rad57 promotes HR at stalled replication forks more than at DNA double-strand breaks (DSB), suggesting that this complex is more efficient at ssDNA gaps and thus, could control the recruitment of TLS polymerases. To address this point, we studied the interplay between Rad55-Rad57 and the TLS polymerases Polζ and Polη following UV radiation. We confirmed that Rad55-Rad57 protects Rad51 filaments from Srs2 dismantling activity but we found that it is also essential for the promotion of UV-induced HR independently of Srs2. In addition, we observed that cell UV sensitivity, but not DSB sensitivity, is synergistically increased when Rad55 and Polζ deletions are combined. Moreover, we found that mutagenesis and HR frequency were increased in rad55∆ mutants and in TLS-deficient cells, respectively. Finally, UV-induced HR was partially restored in Rad55-deficient cells with mutated Polζ or Polη. Overall, our data suggest that the HR and TLS pathways compete for the same ssDNA substrates and that the Rad55-Rad57 complex of Rad51 paralogs prevents the recruitment of TLS polymerases and counterbalances mutagenesis.