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DNA damage tolerance and homologous recombination pathways function to bypass replication-blocking lesions and ensure completion of DNA replication. However, inappropriate activation of these pathways may lead to increased mutagenesis or formation of deleterious recombination intermediates, often leading to cell death or cancer formation in higher organisms. Post-translational modifications of PCNA regulate the choice of repair pathways at replication forks. Its monoubiquitination favors translesion synthesis, while polyubiquitination stimulates template switching. Srs2 helicase binds to small ubiquitin-related modifier (SUMO)-modified PCNA to suppress a subset of Rad51-dependent homologous recombination. Conversely, SUMOylation of Srs2 attenuates its interaction with PCNA. Sgs1 helicase and Mus81 endonuclease are crucial for disentanglement of repair intermediates at the replication fork. Deletion of both genes is lethal and can be rescued by inactivation of Rad51-dependent homologous recombination. Here we show that Saccharomyces cerevisiae Uls1, a member of the Swi2/Snf2 family of ATPases and a SUMO-targeted ubiquitin ligase, physically interacts with both PCNA and Srs2, and promotes Srs2 binding to PCNA by downregulating Srs2-SUMO levels at replication forks. We also identify deletion of ULS1 as a suppressor of mus81Δ sgs1Δ synthetic lethality and hypothesize that uls1Δ mutation results in a partial inactivation of the homologous recombination pathway, detrimental in cells devoid of both Sgs1 and Mus81. We thus propose that Uls1 contributes to the pathway where intermediates generated at replication forks are dismantled by Srs2 bound to SUMO-PCNA. Upon ULS1 deletion, accumulating Srs2-SUMO—unable to bind PCNA—takes part in an alternative PCNA-independent recombination repair salvage pathway(s).

SLX4 assembles a toolkit of endonucleases SLX1, MUS81 and XPF, which is recruited to telomeres via direct interaction of SLX4 with TRF2. Telomeres present an inherent obstacle for DNA replication and repair due to their high propensity to form branched DNA intermediates. Here we provide novel insight into the mechanism and regulation of the SLX4 complex in telomere preservation. SLX4 associates with telomeres throughout the cell cycle, peaking in late S phase and under genotoxic stress. Disruption of SLX4's interaction with TRF2 or SLX1 and SLX1's nuclease activity independently causes telomere fragility, suggesting a requirement of the SLX4 complex for nucleolytic resolution of branched intermediates during telomere replication. Indeed, the SLX1-SLX4 complex processes a variety of telomeric joint molecules in vitro. The nucleolytic activity of SLX1-SLX4 is negatively regulated by telomeric DNA-binding proteins TRF1 and TRF2 and is suppressed by the RecQ helicase BLM in vitro. In vivo, in the presence of functional BLM, telomeric circle formation and telomere sister chromatid exchange, both arising out of nucleolytic processing of telomeric homologous recombination intermediates, are suppressed. We propose that the SLX4-toolkit is a telomere accessory complex that, in conjunction with other telomere maintenance proteins, ensures unhindered, but regulated telomere maintenance.

Fen1 and Mus81–Mms4 are endonucleases involved in the processing of various DNA structural intermediates, and they were shown to have genetic and functional interactions with each other. Here, we show the in vivo significance of the interactions between Mus81 and Rad27 (yeast Fen1). The N-terminal 120 amino-acid (aa) region of Mus81, although entirely dispensable for its catalytic activity, was essential for the abilities of Mus81 to bind to and be stimulated by Rad27. In the absence of SGS1, the mus81Δ120N mutation lacking the N-terminal 120 aa region exhibited synthetic lethality, and the lethality was rescued by deletion of RAD52, a key homologous recombination mediator. These findings, together with the fact that Sgs1 constitutes a redundant pathway with Mus81–Mms4, indicate that the N-terminus-mediated interaction of Mus81 with Rad27 is physiologically important in resolving toxic recombination intermediates. Mutagenic analyses of the N-terminal region identified two distinct motifs, named N21–26 (aa from 21–26) and N108–114 (aa from 108–114) important for the in vitro and in vivo functions of Mus81. Our findings indicate that the N-terminal region of Mus81 acts as a landing pad to interact with Rad27 and that Mus81 and Rad27 work conjointly for efficient removal of various aberrant DNA structures.

Chromosomal double-strand breaks (DSBs) that have only one end with homology to a donor duplex undergo repair by strand invasion followed by replication to the chromosome terminus (break-induced replication, BIR). Using a transformation-based assay system, it was previously shown that BIR could occur by several rounds of strand invasion, DNA synthesis, and dissociation. Here we describe a modification of the transformation-based assay to facilitate detection of switching between donor templates during BIR by genetic selection in diploid yeast. In addition to the expected recovery of template switch products, we found a high frequency of recombination between chromosome homologs during BIR, suggesting transfer of the DSB from the transforming linear DNA to the donor chromosome, initiating secondary recombination events. The frequency of BIR increased in the mph1Δ mutant, but the percentage of template switch events was significantly decreased, revealing an important role for Mph1 in promoting BIR-associated template switching. In addition, we show that the Mus81, Rad1, and Yen1 structure-selective nucleases act redundantly to facilitate BIR.

MUS81 plays important cellular roles in the restart of stalled replication forks, the resolution of recombination intermediates and in telomere length maintenance. Although the actions of MUS81-EME1 have been extensively investigated, MUS81 is the catalytic subunit of two human structure-selective endonucleases, MUS81-EME1 and MUS81-EME2. Little is presently known about the activities of MUS81-EME2. Here, we have purified MUS81-EME2 and compared its activities with MUS81-EME1. We find that MUS81-EME2 is a more active endonuclease than MUS81-EME1 and exhibits broader substrate specificity. Like MUS81-EME1, MUS81-EME2 cleaves 3'-flaps, replication forks and nicked Holliday junctions, and exhibits limited endonuclease activity with intact Holliday junctions. In contrast to MUS81-EME1, however, MUS81-EME2 cuts D-loop recombination intermediates and in so doing disengages the D-loop structure by cleaving the 3'-invading strand. Additionally, MUS81-EME2 acts on 5'-flap structures to cleave off a duplex arm, in reactions that cannot be promoted by MUS81-EME1. These studies suggest that MUS81-EME1 and MUS81-EME2 exhibit similar and yet distinct DNA structure selectivity, indicating that the two MUS81 complexes may promote different nucleolytic cleavage reactions in vivo.

MUS81-EME1 is a DNA endonuclease involved in replication-coupled repair of DNA interstrand cross-links (ICLs). A prevalent hypothetical role of MUS81-EME1 in ICL repair is to unhook the damage by incising the leading strand at the 3' side of an ICL lesion. In this study, we report that purified MUS81-EME1 incises DNA at the 5' side of a psoralen ICL residing in fork structures. Intriguingly, ICL repair protein, Fanconi anemia complementation group A protein (FANCA), greatly enhances MUS81-EME1-mediated ICL incision. On the contrary, FANCA exhibits a two-phase incision regulation when DNA is undamaged or the damage affects only one DNA strand. Studies using truncated FANCA proteins indicate that both the N- and C-moieties of the protein are required for the incision regulation. Using laser-induced psoralen ICL formation in cells, we find that FANCA interacts with and recruits MUS81 to ICL lesions. This report clarifies the incision specificity of MUS81-EME1 on ICL damage and establishes that FANCA regulates the incision activity of MUS81-EME1 in a damage-dependent manner.

The MUS81-EME1 endonuclease maintains metazoan genomic integrity by cleaving branched DNA structures that arise during the resolution of recombination intermediates. In humans, MUS81 also forms a poorly characterized complex with EME2. Here, we identify and determine the structure of a winged helix (WH) domain from human MUS81, which binds DNA. WH domain mutations greatly reduce binding of the isolated domain to DNA and impact on incision activity of MUS81-EME1/EME2 complexes. Deletion of the WH domain reduces the endonuclease activity of both MUS81-EME1 and MUS81-EME2 complexes, and incisions made by MUS81-EME2 are made closer to the junction on substrates containing a downstream duplex, such as fork structures and nicked Holliday junctions. WH domain mutation or deletion in Schizosaccharomyces pombe phenocopies the DNA-damage sensitivity of strains deleted for mus81. Our results indicate an important role for the WH domain in both yeast and human MUS81 complexes.

A key step in meiotic recombination involves the nucleolytic resolution of Holliday junctions to generate crossovers. Although the enzyme that performs this function in human cells is presently unknown, recent studies led to the identification of the XPG-family endonuclease GEN1 that promotes Holliday junction resolution in vitro, suggesting that it may perform a related function in vivo. Here, we show that ectopic expression of GEN1 in fission yeast mus81Δ strains result in Holliday junction resolution and crossover formation during meiosis.

The MUS81 endonuclease complex has been shown to play an important role in the repair of stalled or blocked replication forks and in the processing of meiotic recombination intermediates from yeast to humans. This endonuclease is composed of two subunits, MUS81 and EME1. Surprisingly, unlike other organisms, Arabidopsis (Arabidopsis thaliana) has two EME1 homologs encoded in its genome. AtEME1A and AtEME1B show 63% identity on the protein level. We were able to demonstrate that, after expression in Escherichia coli, each EME1 protein can assemble with the unique AtMUS81 to form a functional endonuclease. Both complexes, AtMUS81-AtEME1A and AtMUS81-AtEME1B, are not only able to cleave 3′-flap structures and nicked Holliday junctions (HJs) but also, with reduced efficiency, intact HJs. While the complexes have the same cleavage patterns with both nicked DNA substrates, slight differences in the processing of intact HJs can be detected. Our results are in line with an involvement of both MUS81-EME1 endonuclease complexes in DNA recombination and repair processes in Arabidopsis.

In budding yeast most Rad51-dependent and -independent recombination depends on Rad52. In contrast, its homologue in fission yeast, Rad22, was assumed to play a less critical role possibly due to functional redundancy with another Rad52-like protein Rti1. We show here that this is not the case. Rad22 like Rad52 plays a central role in recombination being required for both Rhp51-dependent and -independent events. Having established this we proceed to investigate the involvement of the Mus81-Eme1 endonuclease in these pathways. Mus81 plays a relatively minor role in the Rhp51-dependent repair of DNA damage induced by ultraviolet light. In contrast Mus81 has a key role in the Rad22-dependent (Rhp51-independent) repair of damage induced by camptothecin, hydroxyurea and methyl-methanesulfonate. Furthermore, spontaneous intrachromosomal recombination that gives rise to deletion recombinants is impaired in a mus81 mutant. From these data we propose that a Rad22-Mus81-dependent (Rhp51-independent) pathway is an important mechanism for the repair of DNA damage in fission yeast. Consistent with this we show that in vitro Rad22 can promote strand invasion to form a D-loop that can be cleaved by Mus81.

Most models of homologous recombination invoke cleavage of Holliday junctions to explain crossing over. The Mus81·Eme1 endonuclease from fission yeast and humans cleaves Holliday junctions and other branched DNA structures, leaving its physiological substrate uncertain. We report here that Schizosaccharomyces pombe mus81 mutants have normal or elevated frequencies of gene conversion but 20- to 100-fold reduced frequencies of crossing over. Thus, gene conversion and crossing over can be genetically separated, and Mus81 is required for crossing over, supporting the hypothesis that the fission yeast Mus81·Eme1 protein complex resolves Holliday junctions in meiotic cells.

The MMS4 gene of Saccharomyces cerevisiae was originally identified due to its sensitivity to MMS in vegetative cells. Subsequent studies have confirmed a role for MMS4 in DNA metabolism of vegetative cells. In addition, mms4 diploids were observed to sporulate poorly. This work demonstrates that the mms4 sporulation defect is due to triggering of the meiotic recombination checkpoint. Genetic, physical, and cytological analyses suggest that MMS4 functions after the single end invasion step of meiotic recombination. In spo13 diploids, red1, but not mek1, is epistatic to mms4 for sporulation and spore viability, suggesting that MMS4 may be required only when homologs are capable of undergoing synapsis. MMS4 and MUS81 are in the same epistasis group for spore viability, consistent with biochemical data that show that the two proteins function in a complex. In contrast, MMS4 functions independently of MSH5 in the production of viable spores. We propose that MMS4 is required for the processing of specific recombination intermediates during meiosis.