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Updated: Jul 7, 2026
DNAzyme-dependent Analysis of rRNA 2’-O-Methylation
Published on: September 16, 2019
Saccharomyces cerevisiae Mus81-Mms4 is a catalytic, DNA structure-selective endonuclease
Kirk Tevebaugh Ehmsen1, Wolf-Dietrich Heyer
1Section of Microbiology and Section of Molecular and Cellular Biology, University of California Davis, Davis, CA 95616-8665, USA.
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 (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.
Insights
The Mus81-Mms4 endonuclease in yeast supports DNA replication but isn't essential for double-strand break repair. This study characterized its enzymatic activity, finding it acts on various DNA structures but not Holliday junctions in isolation.
Area of Science:
- Molecular Biology
- Enzymology
- DNA Repair
Background:
- Mus81-Mms4/Eme1 is a DNA structure-selective endonuclease involved in DNA replication and recombination.
- Its precise role in double-strand break (DSB) repair in Saccharomyces cerevisiae is context-dependent and not essential.
Purpose of the Study:
- To biochemically characterize the overexpressed and purified Mus81-Mms4 heterodimer from S. cerevisiae.
- To determine the catalytic activity and substrate specificity of Mus81-Mms4 in vitro.
Main Methods:
- Overexpression and purification of the Mus81-Mms4 heterodimer from S. cerevisiae.
- Enzymological characterization including kinetic analysis (kcat, KM) of various DNA structures.
- Analysis of Mus81 and Mms4 phosphorylation states.
Main Results:
- Mus81-Mms4 is catalytically active with distinct substrate preferences (Class I, II, III) including nicked Holliday junctions, 3'-flapped, and replication fork-like structures.
- The enzyme showed low activity on D-loops, partial Holliday junctions, and negligible activity on intact Holliday junctions.
- Phosphorylation of Mus81 and Mms4 occurs constitutively and in response to MMS, but the purified complex showed minimal Holliday junction incision activity.
Conclusions:
- The Mus81-Mms4 heterodimer exhibits specific DNA structure cleavage activity in vitro.
- Its activity is distinct from Holliday junction resolution, suggesting other factors may be involved in this process.
- The in vitro enzymatic characterization provides insights into the functional mechanisms of this key replication-associated endonuclease.
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