Abstract
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.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Cells, Cultured
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DNA / genetics
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DNA / metabolism*
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DNA Polymerase I / genetics
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DNA Polymerase I / metabolism
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DNA Replication / genetics*
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DNA-Binding Proteins / genetics
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DNA-Binding Proteins / metabolism*
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Endonucleases / genetics
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Endonucleases / metabolism*
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Gene Expression Regulation, Enzymologic / genetics
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Gene Expression Regulation, Fungal / genetics
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Macromolecular Substances
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Mutation / genetics
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Nucleic Acid Conformation
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Proteins / metabolism
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Saccharomyces cerevisiae Proteins
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Schizosaccharomyces / enzymology*
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Schizosaccharomyces / genetics
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Schizosaccharomyces pombe Proteins / genetics
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Schizosaccharomyces pombe Proteins / metabolism*
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Substrate Specificity
Substances
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DNA-Binding Proteins
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Macromolecular Substances
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Proteins
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Saccharomyces cerevisiae Proteins
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Schizosaccharomyces pombe Proteins
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xeroderma pigmentosum group F protein
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DNA
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DNA Polymerase I
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Eme1 protein, S pombe
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Endonucleases
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MUS81 protein, S cerevisiae