Functional residues on the surface of the N-terminal domain of yeast Pms1

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Saccharomyces cerevisiae MutLalpha, a heterodimer of Mlh1 and Pms1, is essential for DNA mismatch repair (MMR).
• Pms1 possesses an N-terminal domain (NTD) involved in ATP binding/hydrolysis and DNA binding, and a C-terminal domain (CTD) with endonuclease activity.

Purpose of the Study:

• To elucidate the structural and functional roles of the yeast Pms1 NTD.
• To identify conserved residues involved in DNA binding and protein-protein interactions within the MMR pathway.

Main Methods:

• X-ray crystallography was used to determine the 2.5Å crystal structure of the yeast Pms1 NTD.
• Site-directed mutagenesis was employed to replace conserved surface residues with glutamate.
• In vitro DNA binding assays were performed to assess the impact of mutations on DNA binding affinity.

Main Results:

• The crystal structure of yPms1 NTD revealed similarity to homologous MutL/PMS2 NTDs, including the ATP-binding site.
• Conserved, positively charged surface residues were identified as potential interaction sites.
• Mutations in these residues led to elevated mutation rates in yeast strains, with some mutations decreasing DNA binding affinity.
• Other mutations causing elevated mutation rates did not affect DNA binding, suggesting alternative functional roles.

Conclusions:

• The yPms1 NTD structure provides insights into its function in DNA mismatch repair.
• Specific surface residues are critical for DNA binding and potentially for interactions with other MMR proteins.
• These findings highlight the multifaceted role of Pms1 in maintaining genomic stability.