Claudette L. Doe

Generating Crossovers by Resolution of Nicked Holliday Junctions: A Role for Mus81-Eme1 in Meiosis

The double Holliday junction (dHJ) is generally regarded to be a key intermediate of meiotic recombination, whose resolution is critical for the formation of crossover recombinants. In fission yeast, the Mus81-Eme1 endonuclease has been implicated in resolving dHJs. Consistent with this role, we show that Mus81-Eme1 is required for generating meiotic crossovers. However, purified Mus81-Eme1 prefers to cleave junctions that mimic those formed during the transition from double-strand break to dHJ. Crucially, these junctions are cleaved by Mus81-Eme1 in precisely the right orientation to guarantee the formation of a crossover every time. These data demonstrate how crossovers could arise without forming or resolving dHJs using an enzyme that is widely conserved amongst eukaryotes.

Mus81-Eme1 and Rqh1 Involvement in Processing Stalled and Collapsed Replication Forks

The processing of stalled replication forks and the repair of collapsed replication forks are essential functions in all organisms. In fission yeast DNA junctions at stalled replication forks appear to be processed by either the Rqh1 DNA helicase or Mus81-Eme1 endonuclease. Accordingly, we show that the hypersensitivity to agents that cause replication fork stalling of mus81,eme1, and rqh1 mutants is suppressed by a Holliday junction resolvase (RusA), as is the synthetic lethality of amus81− rqh1− double mutant. Recombinant Mus81-Eme1, purified from Escherichia coli, readily cleaves replication fork structures but cleaves synthetic Holliday junctions relatively poorly in vitro. From these data we propose that Mus81-Eme1 can process stalled replication forks before they have regressed to form a Holliday junction. We also implicate Mus81-Eme1 and Rqh1 in the repair of collapsed replication forks. Here Mus81-Eme1 and Rqh1 seem to function on different substrates because RusA can substitute for Mus81-Eme1 but not Rqh1.

Partial suppression of the fission yeast rqh1− phenotype by expression of a bacterial Holliday junction resolvase

A key stage during homologous recombination is the processing of the Holliday junction, which determines the outcome of the recombination reaction. To dissect the pathways of Holliday junction processing in a eukaryote, we have targeted an Escherichia coli Holliday junction resolvase to the nuclei of fission yeast recombination‐deficient mutants and analysed their phenotypes. The resolvase partially complements the UV and hydroxyurea hypersensitivity and associated aberrant mitoses of an rqh1− mutant. Rqh1 is a member of the RecQ subfamily of DNA helicases that control recombination particularly during S‐phase. Significantly, overexpression of the resolvase in wild‐type cells partly mimics the loss of viability, hyper‐recombination and ‘cut’ phenotype of an rqh1− mutant. These results indicate that Holliday junctions form in wild‐type cells that are normally removed in a non‐recombinogenic way, possibly by Rqh1 catalysing their reverse branch migration. We propose that in the absence of Rqh1, replication fork arrest results in the accumulation of Holliday junctions, which can either impede sister chromatid segregation or lead to the formation of recombinants through Holliday junction resolution.