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Initiation of methyl-directed mismatch repair
1Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710.
The Journal of Biological Chemistry
|June 15, 1992
Summary
The MutH endonuclease in Escherichia coli is activated by DNA mismatches and requires MutS, MutL, and ATP. Activated MutH cleaves unmethylated DNA strands at d(GATC) sites, initiating methyl-directed repair.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Escherichia coli MutH endonuclease has weak d(GATC) endonuclease activity.
- This activity is methylation-dependent.
- DNA repair mechanisms are crucial for genomic stability.
Purpose of the Study:
- To investigate the activation mechanism of MutH endonuclease.
- To understand the role of MutH in methyl-directed DNA repair.
- To elucidate the interaction between DNA mismatches, methylation, and MutH activity.
Main Methods:
- In vitro biochemical assays using purified proteins (MutH, MutS, MutL).
- ATP hydrolysis assays and use of ATP analogs (adenosine-5'-O-(3-thiotriphosphate)).
- DNA substrate manipulation including linear and circular heteroduplexes with varying mismatch and d(GATC) site locations.
Main Results:
- MutH activation requires MutS, MutL, ATP, and Mg2+, and is dependent on DNA mismatches.
- Activation efficiency correlates with mismatch repair efficiency (G-T > G-G > A-C > C-C).
- Activated MutH incises unmethylated d(GATC) sites on either side of the mismatch, with incision occurring 5' to the site on the unmethylated strand.
Conclusions:
- MutH activation is the initiation step of methyl-directed repair.
- MutS binding to a mismatch, followed by ATP-dependent protein translocation, likely triggers MutH activation.
- This process leads to cleavage at d(GATC) sequences, facilitating DNA repair.