Saccharomyces cerevisiae Mus81-Mms4 is a catalytic, DNA structure-selective endonuclease.
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 (k(cat), K(M)) demonstrated that Mus81-Mms4 is catalytically active and identified three substrate classes in vitro. Class I substrates reflect low K(M) (3-7 nM) and high k(cat) ( approximately 1 min(-1)) and include the nicked Holliday junction, 3'-flapped and replication fork-like structures. Class II substrates share low K(M) (1-6 nM) but low k(cat) (< or =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 K(M) ( approximately 30 nM) and low k(cat) (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.
Ehmsen
Kirk Tevebaugh
KT
Section of Microbiology and Section of Molecular and Cellular Biology, University of California Davis, Davis, CA 95616-8665, USA.
Heyer
Wolf-Dietrich
WD
D002021:Buffers; D002384:Catalysis; D004247:DNA / Q000737:chemistry / Q000145:classification / Q000378:metabolism; D045566:DNA, Cruciform / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism*; D045585:Flap Endonucleases; D017353:Gene Deletion; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000737:chemistry; D009153:Mutagens / Q000633:toxicity; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D012965:Sodium Chloride / Q000737:chemistry; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*
D002021:Buffers; D045566:DNA, Cruciform; D004268:DNA-Binding Proteins; D008670:Metals; D009153:Mutagens; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D012965:Sodium Chloride; D004247:DNA; D004706:Endodeoxyribonucleases; D004720:Endonucleases; D045585:Flap Endonucleases; C411624:MUS81 protein, S cerevisiae; C112337:MMS4 protein, S cerevisiae