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MutS mediates heteroduplex loop formation by a translocation mechanism
D J Allen1, A Makhov, M Grilley
1Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA.
Abstract:
Interaction of Escherichia coli MutS and MutL with heteroduplex DNA has been visualized by electron microscopy. In a reaction dependent on ATP hydrolysis, complexes between a MutS dimer and a DNA heteroduplex are converted to protein-stabilized, alpha-shaped loop structures with the mismatch in most cases located within the DNA loop. Loop formation depends on ATP hydrolysis and loop size increases linearly with time at a rate of 370 base pairs/min in phosphate buffer and about 10,000 base pairs/min in the HEPES buffer used for repair assay. These observations suggest a translocation mechanism in which a MutS dimer bound to a mismatch subsequently leaves this site by ATP-dependent tracking or unidimensional movement that is in most cases bidirectional from the mispair. In view of the bidirectional capability of the methyl-directed pathway, this reaction may play a role in determination of heteroduplex orientation. The rate of MutS-mediated DNA loop growth is enhanced by MutL, and when both proteins are present, both are found at the base of alpha-loop structures, and both can remain associated with excision intermediates produced in later stages of the reaction.
Insights
Escherichia coli MutS and MutL proteins form DNA loops at mismatches, driven by ATP hydrolysis. This translocation mechanism, enhanced by MutL, may orient DNA repair pathways.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- DNA repair mechanisms are crucial for maintaining genomic stability.
- Escherichia coli MutS and MutL proteins are key components of the methyl-directed mismatch repair pathway.
Purpose of the Study:
- To visualize the interaction of E. coli MutS and MutL with heteroduplex DNA using electron microscopy.
- To elucidate the mechanism of DNA loop formation and translocation mediated by MutS and MutL.
Main Methods:
- Electron microscopy was used to visualize protein-DNA complexes.
- Biochemical assays were performed to study the kinetics of DNA loop formation in the presence of ATP hydrolysis.
Main Results:
- MutS-DNA complexes form protein-stabilized, alpha-shaped loops with mismatches at the apex.
- Loop formation is dependent on ATP hydrolysis, with loop size increasing linearly over time.
- MutL enhances the rate of MutS-mediated DNA loop growth, and both proteins associate with excision intermediates.
Conclusions:
- The observed loop formation suggests an ATP-dependent translocation mechanism for MutS, potentially bidirectional.
- This mechanism may play a role in determining heteroduplex orientation within the methyl-directed repair pathway.