Interaction of proliferating cell nuclear antigen with PMS2 is required for MutLα activation and function in mismatch...

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• DNA mismatch repair (MMR) is essential for maintaining genomic stability.
• MutLα (MLH1-PMS2 in mammals, MLH1-PMS1 in yeast) is a key endonuclease in MMR.
• Activation of MutLα requires interaction with MutSα/β and DNA-loaded proliferating cell nuclear antigen (PCNA).

Purpose of the Study:

• To investigate the specific interaction between human MutLα and PCNA.
• To identify the molecular determinants of MutLα-PCNA interaction and its functional consequences.
• To assess the role of the PMS2 C-terminal motif in MutLα activation and DNA repair.

Main Methods:

• In vitro biochemical assays to study protein-protein interactions and enzyme activity.
• Site-directed mutagenesis of the PMS2 C-terminal endonuclease domain.
• In vivo mismatch repair assays in yeast models.

Main Results:

• Human PCNA and MutLα form a specific, albeit weak, 1:1 complex.
• PCNA interaction is mediated by the C-terminal endonuclease domain of the PMS2 subunit.
• Mutations in the PMS2 C-terminal ⁷²¹QRLIAP motif abolish PCNA interaction and MutLα activation.
• Mutations in the homologous yeast PMS1 ⁷²³QKLIIP motif impair in vivo mismatch repair.
• Combined mutations exacerbate DNA repair defects.

Conclusions:

• The C-terminal region of PMS2 is critical for MutLα interaction with PCNA.
• This interaction is essential for PCNA-dependent activation of MutLα endonuclease and ATPase activities.
• The identified motif plays a conserved role in DNA mismatch repair across eukaryotes.