Abstract
The budding yeast Srs2 protein possesses 3′ to 5′ DNA helicase activity and channels untimely recombination to post-replication repair by removing Rad51 from ssDNA. However, it also promotes recombination via a synthesis-dependent strand-annealing pathway (SDSA). Furthermore, at the replication fork, Srs2 is required for fork progression and prevents the instability of trinucleotide repeats. To better understand the multiple roles of the Srs2 helicase during these processes, we analysed the ability of Srs2 to bind and unwind various DNA substrates that mimic structures present during DNA replication and recombination. While leading or lagging strands were efficiently unwound, the presence of ssDNA binding protein RPA presented an obstacle for Srs2 translocation. We also tested the preferred directionality of unwinding of various substrates and studied the effect of Rad51 and Mre11 proteins on Srs2 helicase activity. These biochemical results help us understand the possible role of Srs2 in the processing of stalled or blocked replication forks as a part of post-replication repair as well as homologous recombination (HR).
Highlights
The process of homologous recombination (HR) is widespread in nature, as it is present in organisms from bacteria to humans
To better understand the possible role of Srs2 during post replication repair (PRR) and HR, we examined its binding affinity for various DNA structures that may imitate those found during DNA metabolic processes
The DNA substrates were incubated with increasing amounts of Srs2 C, and the reaction mixtures were analysed by electrophoretic mobility shift assay (EMSA)
Summary
The process of homologous recombination (HR) is widespread in nature, as it is present in organisms from bacteria to humans. The first, known as the general DSB repair pathway (DSBR), is characterised by the capture of the second 3 tail and a consequent new round of DNA synthesis and ligation, resulting in the formation of a double Holliday junction (dHJ) This structure can Several helicases have been implicated in the repair of stalled replication forks [1,7]. The deletion of SRS2 confers lethality or strong growth defects when combined with the deletion of several genes involved in DNA repair [7] This synthetic lethality as well as the suppression of the UV sensitivity of post replication repair (PRR) mutants and the hyperecombination phenotype are all alleviated by the deletion of genes from the RAD52 epistasis group [16,18,19,20,21].
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