Phosphorylation of mismatch repair proteins MSH2 and MSH6 affecting MutSalpha mismatch-binding activity

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Mismatch repair (MMR) maintains genomic stability by correcting DNA mismatches, preventing mutations and cancer.
• MMR also mediates genotoxicity and apoptosis in response to DNA lesions like O6-methylguanine.
• While MMR enzymology is well-understood, its regulatory mechanisms remain largely unknown.

Purpose of the Study:

• To investigate the post-translational regulation of MMR, specifically the role of protein phosphorylation.
• To determine if key MMR proteins, MSH2 and MSH6, are subject to phosphorylation and how this affects their function.

Main Methods:

• In vitro phosphorylation assays using protein kinase C, casein kinase II, and protein kinase A.
• In vivo analysis of MSH2 and MSH6 phosphorylation under various conditions (phosphate depletion, kinase inhibition, phosphatase treatment).
• Assessment of MutSalpha complex mismatch-binding activity and nuclear translocation upon mutagen treatment.

Main Results:

• MSH2 and MSH6 proteins, forming the MutSalpha complex, are phosphorylated by protein kinase C and casein kinase II in vitro.
• Phosphorylation of MSH2 and MSH6 occurs in vivo, with MSH6 showing more extensive modification.
• Inhibition or reduction of phosphorylation significantly decreased MutSalpha mismatch-binding activity and prevented methylation-induced nuclear translocation.

Conclusions:

• Protein phosphorylation represents a novel post-translational regulatory mechanism for MMR.
• Phosphorylation of MSH2 and MSH6 enhances MutSalpha mismatch-binding and is essential for its nuclear translocation in response to genotoxic stress.
• This regulation may play a critical role in cellular responses to DNA damage and maintaining genomic integrity.