Mus81, a fission yeast protein related to the XPF subunit of ERCC1-XPF nucleotide excision repair endonuclease, is essential for meiosis and important for coping with stalled replication forks. These processes require resolution of X-shaped DNA structures known as Holliday junctions. We report that Mus81 and an associated protein Eme1 are components of an endonuclease that resolves Holliday junctions into linear duplex products. Mus81 and Eme1 are required during meiosis at a late step of meiotic recombination. The mus81 meiotic defect is rescued by expression of a bacterial Holliday junction resolvase. These findings constitute strong evidence that Mus81 and Eme1 are subunits of a nuclear Holliday junction resolvase.
Cds1, a serine/threonine kinase, enforces the S-M checkpoint in the fission yeast Schizosaccharomyces pombe. Cds1 is required for survival of replicational stress caused by agents that stall replication forks, but how Cds1 performs these functions is largely unknown. Here we report that the forkhead-associated-1 (FHA1) proteindocking domain of Cds1 interacts with Mus81, an evolutionarily conserved damage tolerance protein. Mus81 has an endonuclease homology domain found in the XPF nucleotide excision repair protein. Inactivation of mus81 reveals a unique spectrum of phenotypes. Mus81 enables survival of deoxynucleotide triphosphate starvation, UV radiation, and DNA polymerase impairment. Mus81 is essential in the absence of Bloom's syndrome Rqh1 helicase and is required for productive meiosis. Genetic epistasis studies suggest that Mus81 works with recombination enzymes to properly replicate damaged DNA. Inactivation of Mus81 triggers a checkpoint-dependent delay of mitosis. We propose that Mus81 is involved in the recruitment of Cds1 to aberrant DNA structures where Cds1 modulates the activity of damage tolerance enzymes.Genome integrity is vulnerable during DNA replication. The act of replication can convert a relatively benign single-strand DNA break to a cytotoxic double-strand break. A cyclobutane dimer, formed by UV irradiation, is sufficient to block the progression of DNA polymerases and to cause replication fork collapse (17). To cope with these problems, eukaryotic organisms have developed mechanisms for replicating DNA through and around damage in ways that cause minimal genome instability. Notable among the proteins involved in these processes are lesion bypass polymerases that can replicate through cyclobutane dimers (45). Recombination and double-strand break repair enzymes are also important, participating in the direct repair of DNA structure abnormalities that arise during DNA replication or permitting continued replication around sites of damage (17). These systems, and perhaps others that remain undiscovered, collectively form a genome defense system that allows tolerance of damage during DNA replication.In the fission yeast Schizosaccharomyces pombe, the checkpoint kinase Cds1 is thought to regulate DNA damage tolerance systems (26,28,31). Cds1 is the presumptive homolog of Rad53 in the budding yeast Saccharomyces cerevisiae and Cds1 (also called Chk2) in humans (7,9,16,27). One of its functions is to delay the onset of mitosis when genome replication encounters difficulties (8,26,47). Cds1 enforces this S-M checkpoint by regulating the Cdc25 and Mik1 mitotic control proteins (2,8,18,47). In addition, Cds1 regulates the way in which DNA is replicated under conditions that cause replicational stress. For example, Cds1 is required to slow the replication of DNA that has been damaged by UV irradiation (26,34). A similar function has been ascribed to Rad53 in budding yeast (32). Slowing of DNA replication partly involves the suppression of late-firing replication origins (35, 37). Activation of C...
Functional studies strongly suggest that the Mus81-Eme1 complex resolves Holliday junctions (HJs) in fission yeast, but in vitro it preferentially cleaves flexible three-way branched structures that model replication forks or 3' flaps. Here we report that a nicked HJ is the preferred substrate of endogenous and recombinant Mus81-Eme1. Cleavage occurs specifically on the strand that opposes the nick, resulting in resolution of the structure into linear duplex products. Resolving cuts made by the endogenous Mus81-Eme1 complex on an intact HJ are quasi-simultaneous, indicating that Mus81-Eme1 resolves HJs by a nick and counternick mechanism, with a large rate enhancement of the second cut arising from the flexible nature of the nicked HJ intermediate. Recombinant Mus81-Eme1 is ineffective at making the first cut. We also report that HJs accumulate in a DNA polymerase alpha mutant that lacks Mus81, providing further evidence that the Mus81-Eme1 complex targets HJs in vivo.
Genome integrity is protected by Cds1 (Chk2), a checkpoint kinase that stabilizes arrested replication forks. How Cds1 accomplishes this task is unknown. We report that Cds1 interacts with Rad60, a protein required for recombinational repair in fission yeast. Cds1 activation triggers Rad60 phosphorylation and nuclear delocalization. A Rad60 mutant that inhibits regulation by Cds1 renders cells specifically sensitive to replication fork arrest. Genetic and biochemical studies indicate that Rad60 functions codependently with Smc5 and Smc6, subunits of an SMC (structural maintenance of chromosomes) complex required for recombinational repair. These studies indicate that regulation of Rad60 is an important part of the replication checkpoint response controlled by Cds1. We propose that control of Rad60 regulates recombination events at stalled forks.
Structure-specific DNA endonucleases have critical roles during DNA replication, repair and recombination, yet they also harbor the potential for causing genome instability. Controlling these enzymes may be essential to ensure efficient processing of ad hoc substrates and to prevent random, unscheduled processing of other DNA structures, but it is unknown whether structure-specific endonucleases are regulated in response to DNA damage. Here, we uncover DNA damage-induced activation of Mus81-Eme1 Holliday junction resolvase in fission yeast. This novel regulation requires both Cdc2CDK1 and Rad3ATR-dependent phosphorylations of Eme1. Mus81-Eme1 activation prevents gross chromosomal rearrangements in cells lacking the BLM-related DNA helicase Rqh1. We propose that linking Mus81-Eme1 DNA damaged-induced activation to cell cycle progression ensures efficient resolution of Holliday junctions that escape dissolution by Rqh1-TopIII while preventing unnecessary DNA cleavages.
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