Differential requirement of Srs2 helicase and Rad51 displacement activities in replication of hairpin-forming CAG/CTG repeats

International audienceTrinucleotide repeats are a source of genome instability , causing replication fork stalling, chromosome fragility, and impaired repair. Specialized heli-cases play an important role in unwinding DNA structures to maintain genome stability. The Srs2 heli-case unwinds DNA hairpins, facilitates replication, and prevents repeat instability and fragility. However , since Srs2 is a multifunctional protein with he-licase activity and the ability to displace Rad51 re-combinase, it was unclear which functions were required for its various protective roles. Here, using SRS2 separation-of-function alleles, we show that in the absence of Srs2 recruitment to PCNA or in helicase-deficient mutants, breakage at a CAG/CTG repeat increases. We conclude that Srs2 interaction with PCNA allows the helicase activity to unwind fork-blocking CAG/CTG hairpin structures to prevent breaks. Independently of PCNA binding, Srs2 also displaces Rad51 from nascent strands to prevent recombination-dependent repeat expansions and contractions. By 2D gel electrophoresis, we detect two different kinds of structured interme-diates or joint molecules (JMs). Some JMs are Rad51-independent and exhibit properties of reversed forks, including being processed by the Exo1 nuclease. In addition, in a helicase-deficient mutant, Rad51-dependent JMs are detected, probably corresponding to recombination between sisters. These results clarify the many roles of Srs2 in facilitating replica-tion through fork-blocking hairpin lesions