Differential requirement of Srs2 helicase and Rad51 displacement activities in replication of hairpin-forming CAG/CTG repeats.
Trinucleotide repeats are a source of genome instability, causing replication fork stalling, chromosome fragility, and impaired repair. Specialized helicases play an important role in unwinding DNA structures to maintain genome stability. The Srs2 helicase unwinds DNA hairpins, facilitates replication, and prevents repeat instability and fragility. However, since Srs2 is a multifunctional protein with helicase activity and the ability to displace Rad51 recombinase, 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 intermediates 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 replication through fork-blocking hairpin lesions.
Department of Biology, Tufts University, Medford, MA 02155, USA.
Jennifer H G
JHG
Nguyen
Institut Pasteur, Department Genomes & Genetics, CNRS, UMR3525, Université Pierre et Marie Curie, UFR927, 25 rue du Dr Roux, F-75015 Paris, France.
David
D
Viterbo
Department of Biology, Tufts University, Medford, MA 02155, USA.
Ranjith P
RP
Anand
Department of Biology, Tufts University, Medford, MA 02155, USA.
Lauren
L
Verra
Department of Biology, Tufts University, Medford, MA 02155, USA.
Laura
L
Sloan
Institut Pasteur, Department Genomes & Genetics, CNRS, UMR3525, Université Pierre et Marie Curie, UFR927, 25 rue du Dr Roux, F-75015 Paris, France.
Guy-Franck
GF
Richard
Department of Biology, Tufts University, Medford, MA 02155, USA.
Catherine H
CH
Freudenreich
D000483:Alleles; D002873:Chromosome Fragility; D004265:DNA Helicases / Q000235:genetics* / Q000378:metabolism; D004261:DNA Replication; D004271:DNA, Fungal / Q000235:genetics* / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D005090:Exodeoxyribonucleases / Q000235:genetics / Q000378:metabolism; D016681:Genome, Fungal; D042822:Genomic Instability; D055029:Inverted Repeat Sequences; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D051135:Rad51 Recombinase / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D018911:Trinucleotide Repeats
D004271:DNA, Fungal; D018809:Proliferating Cell Nuclear Antigen; D029701:Saccharomyces cerevisiae Proteins; C061376:SRS2 protein, S cerevisiae; C495151:RAD51 protein, S cerevisiae; D051135:Rad51 Recombinase; D005090:Exodeoxyribonucleases; C028450:exodeoxyribonuclease I; D004265:DNA Helicases