Proliferating cell nuclear antigen facilitates excision in long-patch base excision repair

Area of Science:

• Molecular Biology
• DNA Repair Mechanisms
• Biochemistry

Background:

• Vertebrates utilize two distinct base excision repair (BER) pathways for modified DNA base removal.
• Long-patch BER involves structure-specific endonuclease FEN-1 and proliferating cell nuclear antigen (PCNA)-dependent synthesis.
• The physiological significance of PCNA binding and stimulation of FEN-1 nuclease activity remains unclear.

Purpose of the Study:

• To investigate the importance of the PCNA-FEN-1 interaction in the long-patch BER pathway.
• To determine PCNA's role in facilitating DNA excision mediated by FEN-1.

Main Methods:

• Utilized a reconstituted BER assay system with purified FEN-1.
• Employed PCNA and FEN-1 mutants to assess interaction-dependent functions.
• Manipulated dNTP concentrations to differentiate between excision and synthesis steps.

Main Results:

• Omission of PCNA led to accumulation of pre-excision intermediates, which were resolved upon PCNA addition.
• PCNA was essential for excision intermediate formation when dNTPs were absent.
• A PCNA mutant unable to bind FEN-1 failed to stimulate excision, and a FEN-1 mutant defective in PCNA binding showed reduced activity in BER.

Conclusions:

• PCNA is required for efficient excision during long-patch BER.
• PCNA facilitates the excision step by interacting with FEN-1.
• The PCNA-FEN-1 interaction is physiologically significant for DNA repair.