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.
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