Saccharomyces cerevisiae Mus81-Mms4 is a catalytic, DNA structure-selective endonuclease

Kirk Tevebaugh Ehmsen, Wolf Dietrich Heyer

Research output: Contribution to journalArticle

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Abstract

The DNA structure-selective endonuclease Mus81-Mms4/Eme1 is a context-specific recombination factor that supports DNA replication, but is not essential for DSB repair in Saccharomyces cerevisiae. We overexpressed Mus81-Mms4 in S. cerevisiae, purified the heterodimer to apparent homogeneity, and performed a classical enzymological characterization. Kinetic analysis (kcat, KM) demonstrated that Mus81-Mms4 is catalytically active and identified three substrate classes in vitro. Class I substrates reflect low KM (3-7 nM) and high kcat (∼1 min-1) and include the nicked Holliday junction, 3′-flapped and replication fork-like structures. Class II substrates share low KM (1-6 nM) but low kcat (≤ 0.3 min-1) relative to Class I substrates and include the D-loop and partial Holliday junction. The splayed Y junction defines a class III substrate having high KM (∼30 nM) and low kcat (0.26 min-1). Holliday junctions assembled from oligonucleotides with or without a branch migratable core were negligibly cut in vitro. We found that Mus81 and Mms4 are phosphorylated constitutively and in the presence of the genotoxin MMS. The endogenous complex purified in either modification state is negligibly active on Holliday junctions. Hence, Holliday junction incision activity in vitro cannot be attributed to the Mus81-Mms4 heterodimer in isolation.

Original languageEnglish (US)
Pages (from-to)2182-2195
Number of pages14
JournalNucleic Acids Research
Volume36
Issue number7
DOIs
StatePublished - Apr 2008

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Cruciform DNA
Catalytic DNA
Endonucleases
Saccharomyces cerevisiae
Mutagens
DNA Replication
Oligonucleotides
Genetic Recombination
DNA
In Vitro Techniques

ASJC Scopus subject areas

  • Genetics

Cite this

Saccharomyces cerevisiae Mus81-Mms4 is a catalytic, DNA structure-selective endonuclease. / Ehmsen, Kirk Tevebaugh; Heyer, Wolf Dietrich.

In: Nucleic Acids Research, Vol. 36, No. 7, 04.2008, p. 2182-2195.

Research output: Contribution to journalArticle

Ehmsen, Kirk Tevebaugh ; Heyer, Wolf Dietrich. / Saccharomyces cerevisiae Mus81-Mms4 is a catalytic, DNA structure-selective endonuclease. In: Nucleic Acids Research. 2008 ; Vol. 36, No. 7. pp. 2182-2195.
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