Investigating the role of the Rad51 regulation by Srs2 and Rad54 during DNA replication and repair

DNA carries the information necessary for the continuity of life. However, its integrity is constantly threatened by genotoxic stress exerted by various exogenous and endogenous factors. DNA damage may lead to genetic instability, which can disrupt normal cellular processes and cause cell death. Therefore, multiple mechanisms have evolved to protect the genome integrity. Replication and many DNA repair mechanisms require the formation of single-stranded DNA (ssDNA) intermediates which eventually must be converted back to double-stranded DNA (dsDNA). The results of the experiments performed on the budding yeast Saccharomyces cerevisiae in this study suggest that DNA replication routinely generates ssDNA gaps, most likely at the regions with obstacles for DNA polymerases. These gaps must be filled in postreplicatively to restore the doublestrandedness of DNA, thereby completing the genome duplication. Postreplicative gaps are preferentially repaired by an error-free recombination-based mechanism. This involves the formation of Rad51 nucleofilaments required for the identification of homologous donor sequences which are used as templates for the reconstitution of dsDNA. The elimination of two Rad51 regulators, Srs2 and Rad54, causes the death of Saccharomyces cerevisiae cells. In srs2Δ rad54Δ double mutants, postreplicative gaps cannot be resolved by recombination as Rad54 is necessary for this type of repair. The alternative pathway to restore doublestrandedness involves a simple filling of ssDNA gaps by DNA polymerases. However, although necessary for the establishment of recombination intermediates, Rad51 filaments can hinder the recruitment and loading of DNA synthesis machinery at DNA damage sites. Srs2 helicase can promote damage-associated DNA synthesis by disassembling Rad51 nucleofilaments formed on ssDNA. It was demonstrated in this study that Rad54 translocase capable of removing Rad51 from dsDNA can facilitate the DNA synthesis during DNA repair along with Srs2. Further analysis has revealed that the activities of Srs2 and Rad54 in the said process are mostly complementary rather than redundant. This is most likely because the two proteins work on different substrates – Rad51 filaments formed on ssDNA and dsDNA respectively. The srs2Δ rad54Δ mutants were found to accumulate chromatin-bound Rad51 after a single round of DNA replication suggesting that Rad51 filaments cannot be effectively disassembled in these cells. Furthermore, extensive DNA loss was observed after the double mutants attempted to divide and entered the second round of DNA replication. The elimination of the DNA end resection nuclease Exo1 rescued the viability of the srs2Δ rad54Δ cells. The nucleolytic processing of ssDNA gaps is known to be required for an efficient formation of Rad51 nucleofilaments. Thus, the excessive expansion of ssDNA gaps by Exo1 and the failure to repair them due to the inability to disassemble Rad51 nucleofilaments is the likely explanation of the DNA loss and cell death in the absence of both Srs2 and Rad54. Overall, this study provides evidence that the disassembly of Rad51 nucleofilaments by Srs2 and/or Rad54 is necessary to enable the filling of postreplicative ssDNA gaps by DNA polymerases and is likely required for the routine genome maintenance