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Cleavage mechanism of human Mus81-Eme1 acting on Holliday-junction structures.

ABSTRACT: Recombination-mediated repair plays a central role in maintaining genomic integrity during DNA replication. The human Mus81-Eme1 endonuclease is involved in recombination repair, but the exact structures it acts on in vivo are not known. Using kinetic and enzymatic analysis of highly purified recombinant enzyme, we find that Mus81-Eme1 catalyzes coordinate bilateral cleavage of model Holliday-junction structures. Using a self-limiting, cruciform-containing substrate, we demonstrate that bilateral cleavage occurs sequentially within the lifetime of the enzyme-substrate complex. Coordinate bilateral cleavage is promoted by the highly cooperative nature of the enzyme and results in symmetrical cleavage of a cruciform structure, thus, Mus81-Eme1 can ensure coordinate, bilateral cleavage of Holliday junction-like structures.

SUBMITTER: Taylor ER

PROVIDER: S-EPMC2268786 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Regulation of Mus81-Eme1 Holliday junction resolvase in response to DNA damage.

Project description:Structure-specific DNA endonucleases have critical roles during DNA replication, repair and recombination, yet they also have 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 new regulation requires both Cdc2(CDK1)- and Rad3(ATR)-dependent phosphorylation 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 damage-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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Cooperative control of holliday junction resolution and DNA repair by the SLX1 and MUS81-EME1 nucleases.

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Human Rad54 protein stimulates human Mus81-Eme1 endonuclease.

Project description:Rad54, a key protein of homologous recombination, physically interacts with a DNA structure-specific endonuclease, Mus81-Eme1. Genetic data indicate that Mus81-Eme1 and Rad54 might function together in the repair of damaged DNA. In vitro, Rad54 promotes branch migration of Holliday junctions, whereas the Mus81-Eme1 complex resolves DNA junctions by endonucleolytic cleavage. Here, we show that human Rad54 stimulates Mus81-Eme1 endonuclease activity on various Holliday junction-like intermediates. This stimulation is the product of specific interactions between the human Rad54 (hRad54) and Mus81 proteins, considering that Saccharomyces cerevisiae Rad54 protein does not stimulate human Mus81-Eme1 endonuclease activity. Stimulation of Mus81-Eme1 cleavage activity depends on formation of specific Rad54 complexes on DNA substrates occurring in the presence of ATP and, to a smaller extent, of other nucleotide cofactors. Thus, our results demonstrate a functional link between the branch migration activity of hRad54 and the structure-specific endonuclease activity of hMus81-Eme1, suggesting that the Rad54 and Mus81-Eme1 proteins may cooperate in the processing of Holliday junction-like intermediates during homologous recombination or DNA repair.

| S-EPMC2587595 | biostudies-literature

Overlapping roles for Yen1 and Mus81 in cellular Holliday junction processing.

Project description:Eukaryotic Holliday junction (HJ) resolvases have attracted much attention recently with the identification of at least three distinct proteins that can cleave model HJs in vitro. However, the specific DNA structure(s) that these proteins act upon in the cell is unknown. Here, we describe a system in budding yeast to directly and quantitatively monitor in vivo HJ resolution. We found that Yen1 acts redundantly with Mus81, but not Slx1, to resolve a model HJ in vivo. This functional overlap specifically extends to the repair/bypass of lesions that impede the progression of replication forks but not to the repair of double-strand breaks induced by ionizing radiation. Together, these results suggest a direct role for Yen1 in the response to DNA damage and implicate overlapping HJ resolution functions of Yen1 with Mus81 during replication fork repair.

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The human Holliday junction resolvase GEN1 rescues the meiotic phenotype of a Schizosaccharomyces pombe mus81 mutant.

Project description: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 mus81Delta strains results in Holliday junction resolution and crossover formation during meiosis.

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Mus81 cleavage of Holliday junctions: a failsafe for processing meiotic recombination intermediates?

Project description:The Holliday junction (HJ) is a central intermediate of homologous recombination. Its cleavage is critical for the formation of crossover recombinants during meiosis, which in turn helps to establish chiasmata and promote genetic diversity. Enzymes that cleave HJs, called HJ resolvases, have been identified in all domains of life except eukaryotic nuclei. Controversially, the Mus81-Eme1 endonuclease has been proposed to be an example of a eukaryotic nuclear resolvase. However, hitherto little or no HJ cleavage has been detected in recombinant preparations of Mus81-Eme1. Here, we report the purification of active forms of recombinant Schizosaccharomyces pombe Mus81-Eme1 and Saccharomyces cerevisiae Mus81-Mms4, which display robust HJ cleavage in vitro, which, in the case of Mus81-Eme1, is as good as the archetypal HJ resolvase RuvC in single turnover kinetic analysis. We also present genetic evidence that suggests that this activity might be utilised as a back-up to Mus81-Eme1's main activity of cleaving nicked HJs during meiosis in S. pombe.

| S-EPMC1847671 | biostudies-literature

Cleavage of stalled forks by fission yeast Mus81/Eme1 in absence of DNA replication checkpoint.

Project description:During replication arrest, the DNA replication checkpoint plays a crucial role in the stabilization of the replisome at stalled forks, thus preventing the collapse of active forks and the formation of aberrant DNA structures. How this checkpoint acts to preserve the integrity of replication structures at stalled fork is poorly understood. In Schizosaccharomyces pombe, the DNA replication checkpoint kinase Cds1 negatively regulates the structure-specific endonuclease Mus81/Eme1 to preserve genomic integrity when replication is perturbed. Here, we report that, in response to hydroxyurea (HU) treatment, the replication checkpoint prevents S-phase-specific DNA breakage resulting from Mus81 nuclease activity. However, loss of Mus81 regulation by Cds1 is not sufficient to produce HU-induced DNA breaks. Our results suggest that unscheduled cleavage of stalled forks by Mus81 is permitted when the replisome is not stabilized by the replication checkpoint. We also show that HU-induced DNA breaks are partially dependent on the Rqh1 helicase, the fission yeast homologue of BLM, but are independent of its helicase activity. This suggests that efficient cleavage of stalled forks by Mus81 requires Rqh1. Finally, we identified an interplay between Mus81 activity at stalled forks and the Chk1-dependent DNA damage checkpoint during S-phase when replication forks have collapsed.

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Crystal structures of the structure-selective nuclease Mus81-Eme1 bound to flap DNA substrates.

Project description:The Mus81-Eme1 complex is a structure-selective endonuclease with a critical role in the resolution of recombination intermediates during DNA repair after interstrand cross-links, replication fork collapse, or double-strand breaks. To explain the molecular basis of 3' flap substrate recognition and cleavage mechanism by Mus81-Eme1, we determined crystal structures of human Mus81-Eme1 bound to various flap DNA substrates. Mus81-Eme1 undergoes gross substrate-induced conformational changes that reveal two key features: (i) a hydrophobic wedge of Mus81 that separates pre- and post-nick duplex DNA and (ii) a "5' end binding pocket" that hosts the 5' nicked end of post-nick DNA. These features are crucial for comprehensive protein-DNA interaction, sharp bending of the 3' flap DNA substrate, and incision strand placement at the active site. While Mus81-Eme1 unexpectedly shares several common features with members of the 5' flap nuclease family, the combined structural, biochemical, and biophysical analyses explain why Mus81-Eme1 preferentially cleaves 3' flap DNA substrates with 5' nicked ends.

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Crystal structure of the Mus81-Eme1 complex.

Project description:The Mus81-Eme1 complex is a structure-specific endonuclease that plays an important role in rescuing stalled replication forks and resolving the meiotic recombination intermediates in eukaryotes. We have determined the crystal structure of the Mus81-Eme1 complex. Both Mus81 and Eme1 consist of a central nuclease domain, two repeats of the helix-hairpin-helix (HhH) motif at their C-terminal region, and a linker helix. While each domain structure resembles archaeal XPF homologs, the overall structure is significantly different from those due to the structure of a linker helix. We show that a flexible intradomain linker that formed with 36 residues in the nuclease domain of Eme1 is essential for the recognition of DNA. We identified several basic residues lining the outer surface of the active site cleft of Mus81 that are involved in the interaction with a flexible arm of a nicked Holliday junction (HJ). These interactions might contribute to the optimal positioning of the opposite junction across the nick into the catalytic site, which provided the basis for the "nick and counternick" mechanism of Mus81-Eme1 and for the nicked HJ to be the favored in vitro substrate of this enzyme.

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