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Detection of high-affinity and sliding clamp modes for MSH2-MSH6 by single-molecule unzipping force analysis
Jingjing Jiang1, Lu Bai, Jennifer A Surtees
1Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853, USA.
Abstract:
Mismatch repair (MMR) is initiated by MutS family proteins (MSH) that recognize DNA mismatches and recruit downstream repair factors. We used a single-molecule DNA-unzipping assay to probe interactions between S. cerevisiae MSH2-MSH6 and a variety of DNA mismatch substrates. This work revealed a high-specificity binding state of MSH proteins for mismatch DNA that was not observed in bulk assays and allowed us to measure the affinity of MSH2-MSH6 for mismatch DNA as well as its footprint on DNA surrounding the mismatch site. Unzipping analysis with mismatch substrates containing an end blocked by lac repressor allowed us to identify MSH proteins present on DNA between the mismatch and the block, presumably in an ATP-dependent sliding clamp mode. These studies provide a high-resolution approach to study MSH interactions with DNA mismatches and supply evidence to support and refute different models proposed for initiation steps in MMR.
Insights
Single-molecule DNA unzipping reveals high-specificity binding of MutS homolog (MSH) proteins to DNA mismatches. This high-resolution method quantifies MSH2-MSH6 affinity and DNA footprint, advancing mismatch repair (MMR) models.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- DNA mismatch repair (MMR) is crucial for genomic stability.
- MutS homolog (MSH) proteins initiate MMR by recognizing DNA mismatches.
- Existing bulk assays lack the resolution to fully characterize MSH-DNA interactions.
Purpose of the Study:
- To investigate the high-specificity binding of Saccharomyces cerevisiae MSH2-MSH6 to DNA mismatches.
- To quantify the binding affinity and DNA footprint of MSH2-MSH6 using a single-molecule approach.
- To explore the potential sliding clamp mechanism of MSH proteins during MMR initiation.
Main Methods:
- Single-molecule DNA-unzipping assay.
- Utilized various DNA mismatch substrates, including those with a blocked end (lac repressor).
- Measured protein-DNA interactions at high resolution.
Main Results:
- Identified a high-specificity binding state of MSH proteins for DNA mismatches, not detectable by bulk assays.
- Quantified the binding affinity and determined the DNA footprint of MSH2-MSH6 around mismatches.
- Provided evidence for ATP-dependent sliding of MSH proteins on DNA between the mismatch and a blocked end.
Conclusions:
- Single-molecule DNA unzipping offers a high-resolution method to study MSH-DNA interactions in MMR.
- The findings support and refine models for the initiation steps of DNA mismatch repair.
- This approach can help differentiate between proposed mechanisms for MMR initiation.
