Abstract
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.
Highlights
The XPF family of eukaryotic DNA junction endonucleases plays crucial roles in maintaining genomic stability by functioning in multiple DNA processing pathways [1]
Our results for MUS81-EME1 contrasts with studies where the endonuclease activity of the complex was not affected by the removal of the N-termini of both MUS81 and EME1 [4,25,26]
We have used the longer EME1583 transcript variant of EME1, isolated by Ciccia et al [2], which has a 13 amino acid insert after residue 371 residing within the 36R linker region identified by Chang et al [4] and shown to be important for DNA binding and endonuclease activity
Summary
The XPF family of eukaryotic DNA junction endonucleases plays crucial roles in maintaining genomic stability by functioning in multiple DNA processing pathways [1]. These include XPF-ERCC1, MUS81-EME1 and the largely uncharacterized MUS81-EME2 complex as well as FANCMFAAP24, this complex has no demonstrable nuclease activity to date [1,2]. The MUS81 endonuclease, when associated with the non-catalytic partner EME1, is able to efficiently cleave a variety of three- and four-way junctions containing a duplex downstream from a nick in vitro [7]. These structures include forks, 30 flaps, nicked Holliday junctions and D-loops [4,8]. Such MUS81-EME1 substrates can form during mitosis and fission yeast meiosis and during processing of damaged replication forks
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