Cleavage of model replication forks by fission yeast Mus81-Eme1 and budding yeast Mus81-Mms4.

The blockage of replication forks can result in the disassembly of the replicative apparatus and reversal of the fork to form a DNA junction that must be processed in order for replication to restart and sister chromatids to segregate at mitosis. Fission yeast Mus81-Eme1 and budding yeast Mus81-Mms4 are endonucleases that have been implicated in the processing of aberrant DNA junctions formed at stalled replication forks. Here we have investigated the activity of purified Mus81-Eme1 and Mus81-Mms4 on substrates that resemble DNA junctions that are expected to form when a replication fork reverses. Both enzymes cleave Holliday junctions and substrates that resemble normal replication forks poorly or not at all. However, forks where the equivalents of either both the leading and lagging strands or just the lagging strand are juxtaposed at the junction point, or where either the leading or lagging strand has been unwound to produce a fork with a single-stranded tail, are cleaved well. Cleavage sites map predominantly between 3 and 6 bp 5' of the junction point. For most substrates the leading strand template is cleaved. The sole exception is a fork with a 5' single-stranded tail, which is cleaved in the lagging strand template

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

The Holliday junction resolvase SpCCE1 prevents mitochondrial DNA aggregation in Schizosaccharomyces pombe.

SpCCE1 (YDC2) from Schizosaccharomyces pombe is a DNA structure-specific endonuclease that resolves Holliday junctions in vitro. To investigate the in vivo function of SpCCE1 we made an Spcce1:ura4+ insertion mutant strain. This strain is viable and, despite being devoid of the Holliday junction resolvase activity that is readily detected in fractionated extracts from wild-type cells, exhibits normal levels of UV sensitivity and spontaneous or UV-induced mitotic recombination. In accordance with the absence of a nuclear phenotype, we show by fluorescence microscopy that a SpCCE1-GFP fusion localises exclusively to the mitochondria of S. pombe. In Saccharomyces cerevisiae the homologue of SpCCE1, CCE1, is known to function in the mitochondria where its role appears to be to remove recombination junctions and thus facilitate mitochondrial DNA segregation. A similar function can probably be attributed to SpCCE1 in S. pombe, since the majority of mitochondrial DNA from the Spcce1::ura4- strain is in an aggregated form apparently due to extensive interlinking of DNA molecules by recombination junctions. Surprisingly, this marked effect on the conformation of mitochondrial DNA results in little or no effect on proliferation or viability of the Spcce1::ura4+ strain. Possible explanations are discussed

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