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Updated: Jun 4, 2025
Application of Stopped-flow Kinetics Methods to Investigate the Mechanism of Action of a DNA Repair Protein
Published on: March 31, 2010
Mlh1-Pms1 ATPase activity is regulated distinctly by self-generated nicks and strand discrimination signals in
Jonathan M Piscitelli1, Scott J Witte1, Yasmine S Sakinejad1
1Department of Chemistry, Temple University, 1901 N. 13th St. Philadelphia, PA 19122, USA.
Abstract:
In eukaryotic post-replicative mismatch repair, MutS homolog complexes detect mismatches and in the major eukaryotic pathway, recruit Mlh1-Pms1/MLH1-PMS2 (yeast/human) complexes, which nick the newly replicated DNA strand upon activation by the replication processivity clamp, PCNA. This incision enables mismatch removal and DNA repair. Beyond its endonuclease role, Mlh1-Pms1/MLH1-PMS2 also has ATPase activity, which genetic studies suggest is essential for mismatch repair, although its precise regulatory role on DNA remains unclear. Here, we use an ATP-binding and hydrolysis-deficient yeast Mlh1-Pms1 variant to show that ATP hydrolysis promotes disengagement from Mlh1-Pms1-generated nicks, with hydrolysis in the Mlh1 subunit driving this activity. Our data suggest that the ATPase-deficient variant becomes trapped on its own endonuclease product, suggesting a mechanistic explanation for observations in genetic experiments. Additionally, we observed that Mlh1-Pms1 selectively protects DNA from exonuclease degradation at pre-existing nicks, which may act as strand discrimination signals in mismatch repair. Together, our findings suggest that Mlh1-Pms1 exhibits distinct behaviors on its own endonuclease products versus substrates with pre-existing nicks, supporting two distinct modes of action during DNA mismatch repair.
Insights
ATP hydrolysis by the Mlh1-Pms1 complex is crucial for DNA mismatch repair. This process allows the complex to disengage from DNA nicks, preventing it from becoming trapped and ensuring efficient repair.
Area of Science:
- Molecular Biology
- DNA Repair Mechanisms
- Biochemistry
Background:
- Eukaryotic post-replicative mismatch repair involves MutS homolog complexes and Mlh1-Pms1/MLH1-PMS2 to correct errors.
- Mlh1-Pms1/MLH1-PMS2 possesses endonuclease activity and ATPase activity, with the latter's role in DNA repair being unclear.
- PCNA (proliferating cell nuclear antigen) activates the nicking activity of Mlh1-Pms1/MLH1-PMS2.
Purpose of the Study:
- To investigate the role of ATP hydrolysis in the function of the Mlh1-Pms1 complex during DNA mismatch repair.
- To elucidate the mechanism by which Mlh1-Pms1 interacts with DNA nicks.
- To understand how Mlh1-Pms1 distinguishes between self-generated nicks and pre-existing nicks.
Main Methods:
- Utilized an ATP-binding and hydrolysis-deficient yeast Mlh1-Pms1 variant.
- Assessed the disengagement of the Mlh1-Pms1 complex from DNA nicks.
- Investigated the protective effect of Mlh1-Pms1 on DNA nicks against exonuclease degradation.
Main Results:
- ATP hydrolysis, specifically in the Mlh1 subunit, promotes the disengagement of Mlh1-Pms1 from nicks it generates.
- An ATPase-deficient Mlh1-Pms1 variant gets trapped on its own endonuclease products.
- Mlh1-Pms1 protects DNA at pre-existing nicks from exonuclease degradation, potentially aiding strand discrimination.
Conclusions:
- Mlh1-Pms1's ATPase activity is essential for its release from DNA nicks, explaining genetic observations.
- The complex exhibits distinct binding behaviors at self-generated nicks versus pre-existing nicks.
- These findings support two distinct modes of action for Mlh1-Pms1 in DNA mismatch repair.
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