Related Experiment Video
Updated: Aug 20, 2026
Efficient Sporulation of Saccharomyces cerevisiae in a 96 Multiwell Format
Published on: September 17, 2016
Substrate specificity of the Saccharomyces cerevisiae Mus81-Mms4 endonuclease
William M Fricke1, Suzanne A Bastin-Shanower, Steven J Brill
1Department of Molecular Biology and Biochemistry, Rutgers University, 679 Hoes Lane, Piscataway, NJ 08854, USA.
Abstract:
Mus81-Mms4/Eme1 is a conserved structure-specific endonuclease that functions in mitotic and meiotic recombination. It has been difficult to identify a single preferred substrate of this nuclease because it is active on a variety of DNA structures. In addition, it has been suggested that the specificity of the recombinant protein may differ from that of the native enzyme. Here, we addressed these issues with respect to Mus81-Mms4 from S. cerevisiae. At low substrate concentrations, Mus81-Mms4 was active on any substrate containing a free end adjacent to the branchpoint. This includes 3'-flap (3'F), regressed leading strand replication fork (RLe), regressed lagging strand replication fork (RLa), and nicked Holliday junction (nHJ) substrates. Kinetic analysis was used to quantitate differences between substrates. High Kcat/Km values were obtained only for substrates with a 5'-end near the branchpoint (i.e., 3'F, RLe, and nHJ); 10-fold lower values were obtained for nicked duplex (nD) and RLa substrates. Substrates lacking any free ends at the branch point generated Kcat/Km values that were four orders of magnitude lower than those of the preferred substrates. Native Mus81-Mms4 was partially purified from yeast cells and found to retain its preference for 3'F over intact HJ substrates. Taken together, these results narrow the range of optimal substrates for Mus81-Mms4 and indicate that, at least for S. cerevisae, the native and recombinant enzymes display similar substrate specificities.
Insights
The Mus81-Mms4 endonuclease prefers DNA structures with a free end near the branchpoint, such as 3'-flap and nicked Holliday junctions. Both native and recombinant Mus81-Mms4 enzymes from yeast show similar substrate preferences.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Mus81-Mms4/Eme1 is a crucial structure-specific endonuclease involved in DNA repair and genome stability during mitosis and meiosis.
- Identifying the precise substrate specificity of Mus81-Mms4 has been challenging due to its activity on diverse DNA structures.
- Potential differences between recombinant and native Mus81-Mms4 enzyme specificities require investigation.
Purpose of the Study:
- To determine the preferred DNA substrates of the Mus81-Mms4 endonuclease from Saccharomyces cerevisiae.
- To compare the substrate specificity of recombinant Mus81-Mms4 with the native enzyme purified from yeast cells.
Main Methods:
- Enzyme kinetics were employed to quantify the activity of Mus81-Mms4 on various DNA substrates, including 3 ac-flap (3 acF), regressed leading (RLe) and lagging (RLa) strand replication forks, nicked Holliday junctions (nHJ), and nicked duplex (nD) DNA.
- Native Mus81-Mms4 was partially purified from S. cerevisiae.
- Comparative analysis of catalytic efficiency (Kcat/Km) across different substrate types.
Main Results:
- Mus81-Mms4 exhibited activity on substrates with a free end adjacent to the branchpoint, including 3 acF, RLe, RLa, and nHJ.
- Optimal substrate recognition, indicated by high Kcat/Km values, was observed for substrates with a 5 ac-end near the branchpoint (3 acF, RLe, nHJ).
- Substrates lacking free ends at the branchpoint showed significantly lower activity, and native Mus81-Mms4 retained its preference for 3 acF over intact Holliday junctions.
Conclusions:
- The study defines a narrower range of optimal substrates for Mus81-Mms4, highlighting the importance of a free end near the branchpoint.
- Results indicate that both native and recombinant Mus81-Mms4 enzymes from S. cerevisiae possess similar substrate specificities.
- This clarifies the enzymatic properties of Mus81-Mms4, contributing to understanding its role in DNA recombination and genome maintenance.
Related Concept Videos
01:11Restriction Enzymes
The host bacteria protect their own genomic DNA from these enzymes by methylating these sites. Some...
01:20Mismatch Repair
The Mutator Protein Family Plays a Key Role in DNA Mismatch Repair
The human genome has more than 3 billion base pairs of DNA per cell. Prior to cell division, that vast amount of genetic...