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Sharp kinking of a coiled-coil in MutS allows DNA binding and release
Doreth Bhairosing-Kok1, Flora S Groothuizen1, Alexander Fish1
1Division of Biochemistry and Oncode Institute, Netherlands Cancer Institute, 1066 CX Amsterdam, the Netherlands.
Nucleic Acids Research
|August 3, 2019
Summary
The DNA mismatch repair (MMR) protein MutS uses flexible domains to bind DNA. This study reveals a specific kinking mechanism in MutS lever domains crucial for DNA loading and MMR function.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- DNA mismatch repair (MMR) is essential for genomic stability.
- The MutS protein, a key component of MMR, binds to DNA mismatches using its lever and clamp domains.
- Previous research suggested flexibility in MutS domains, but its functional significance remained unclear.
Purpose of the Study:
- To investigate the structural basis of DNA-free MutS flexibility.
- To determine the role of MutS domain flexibility in DNA binding and MMR function.
- To elucidate the mechanism of DNA loading by MutS.
Main Methods:
- X-ray crystallography to determine the structure of DNA-free Escherichia coli MutS (apo-structure).
- Site-directed mutagenesis to alter specific hinge regions in the MutS coiled-coil lever domains.
- DNA binding assays to quantify the effect of mutations on MutS-DNA interaction affinity.
Main Results:
- A novel crystal structure of DNA-free E. coli MutS revealed repositioned clamp domains due to kinking in the lever domain's coiled-coil region.
- Mutations disrupting the coiled-coil kinking diminished DNA binding affinity.
- Mutations stabilizing the coiled-coil structure enhanced DNA binding.
Conclusions:
- Site-specific kinking in the MutS coiled-coil lever domain acts as a hinge, playing a critical role in DNA loading.
- This structural flexibility is essential for the functional mechanism of MutS as an ABC-ATPase in DNA repair.
- Understanding MutS structural dynamics provides insights into the precise mechanism of DNA mismatch recognition and repair initiation.
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