In eukaryotic flap endonuclease 1, the C terminus is essential for substrate binding

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Flap endonuclease 1 (Fen1) is a structure-specific metallonuclease vital for DNA replication and repair.
• Fen1 interacts with proliferating cell nuclear antigen (PCNA), which stimulates its enzymatic activity in vitro.
• The PCNA interaction site on Fen1 is near its C terminus, adjacent to a conserved basic region in eukaryotes.

Purpose of the Study:

• To investigate the functional roles of the C-terminal PCNA interaction motif and adjacent basic region in human Fen1.
• To analyze the impact of deleting these regions on Fen1's enzymatic activity, PCNA interaction, and substrate binding.

Main Methods:

• Construction and analysis of two human Fen1 deletion mutants.
• Enzymatic activity assays.
• Electrophoretic mobility shift assays (EMSA) to assess substrate binding.
• Analysis of protein-PCNA interactions.

Main Results:

• Deletion of the C-terminal basic region did not abolish PCNA interaction.
• This deletion mutant exhibited significantly reduced enzymatic activity.
• EMSA revealed a severe defect in substrate binding for the C-terminal deletion mutant.
• The PCNA interaction motif deletion mutant was not explicitly detailed in its results, but the focus was on the C-terminal region.

Conclusions:

• The C terminus of eukaryotic Fen1 contains at least two functionally distinct regions.
• These regions, including the basic C-terminal region, play crucial roles in regulating Fen1's enzymatic activity and substrate binding.
• These findings suggest a complex regulatory mechanism involving the Fen1 C terminus for DNA metabolic processes.