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Application of Stopped-flow Kinetics Methods to Investigate the Mechanism of Action of a DNA Repair Protein
Published on: March 31, 2010
Formation of a DNA mismatch repair complex mediated by ATP
Tassadite Selmane1, Mark J Schofield, Sunil Nayak
1Genetics and Biochemistry Branch, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-1810, USA.
Abstract:
The mismatch repair proteins, MutS and MutL, interact in a DNA mismatch and ATP-dependent manner to activate downstream events in repair. Here, we assess the role of ATP binding and hydrolysis in mismatch recognition by MutS and the formation of a ternary complex involving MutS and MutL bound to a mismatched DNA. We show that ATP reduces the affinity of MutS for mismatched DNA and that the modulation of DNA binding affinity by nucleotide is even more pronounced for MutS E694A, a protein that binds ATP but is defective for ATP hydrolysis. Despite the ATP hydrolysis defect, E694A, like WT MutS, undergoes rapid, ATP-dependent dissociation from a DNA mismatch. Furthermore, MutS E694A retains the ability to interact with MutL on mismatched DNA. The recruitment of MutL to a mismatched DNA by MutS is also observed for two mutant MutL proteins, E29A, defective for ATP hydrolysis, and R266A, defective for DNA binding. These results suggest that ATP binding in the absence of hydrolysis is sufficient to trigger formation of a MutS sliding clamp. However, recruitment of MutL results in the formation of a dynamic ternary complex that we propose is the intermediate that signals subsequent repair steps requiring ATP hydrolysis.
Insights
ATP binding, not hydrolysis, allows MutS to form a sliding clamp on DNA. However, MutL recruitment creates a dynamic complex essential for DNA repair signaling.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- DNA mismatch repair (MMR) is crucial for genomic stability.
- MutS and MutL proteins are key components of the MMR pathway.
- Their interaction is ATP-dependent, but the precise roles of ATP binding and hydrolysis are not fully understood.
Purpose of the Study:
- To investigate the specific roles of ATP binding and hydrolysis in MutS-DNA interaction and MutS-MutL complex formation.
- To elucidate the mechanism of MMR initiation.
Main Methods:
- Site-directed mutagenesis of MutS (E694A) and MutL (E29A, R266A) proteins.
- Analysis of protein-DNA binding affinities using nucleotide binding and hydrolysis assays.
- Investigation of protein-protein interactions in vitro.
Main Results:
- ATP binding reduces MutS affinity for mismatched DNA, an effect amplified in MutS E694A (defective in hydrolysis).
- MutS E694A dissociates rapidly from DNA upon ATP binding, similar to wild-type MutS.
- MutS E694A can still recruit MutL, even when MutL is defective in hydrolysis (E29A) or DNA binding (R266A).
Conclusions:
- ATP binding alone, without hydrolysis, is sufficient for MutS to form a sliding clamp on DNA.
- MutL recruitment by MutS leads to a dynamic ternary complex, likely signaling downstream repair events that require ATP hydrolysis.
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