Novel function of the flap endonuclease 1 complex in processing stalled DNA replication forks

Area of Science:

• Molecular Biology
• DNA Replication
• DNA Repair

Background:

• Restarting stalled DNA replication forks is essential for genomic stability.
• The break-induced recombination pathway involves creating DNA double-stranded breaks (DSBs) at stalled forks.
• The nucleases responsible for generating DSBs from single-stranded DNA gaps at stalled forks in eukaryotes remain largely unidentified.

Purpose of the Study:

• To identify the nucleases involved in generating DSBs at stalled replication forks.
• To characterize the novel gap endonuclease (GEN) activity of human flap endonuclease 1 (FEN-1).
• To elucidate the role of FEN-1 in resolving stalled replication forks.

Main Methods:

• Biochemical assays to identify endonuclease activity.
• Protein-protein interaction studies (co-immunoprecipitation).
• Functional complementation assays in a yeast FEN-1 null mutant.

Main Results:

• Human flap endonuclease 1 (FEN-1) exhibits previously unrecognized gap endonuclease (GEN) activity.
• FEN-1 specifically interacts with Werner syndrome protein upon replication arrest to cleave stalled forks.
• Replication protein A enhances FEN-1's interaction with DNA bubble structures.
• Wild-type human FEN-1, but not a GEN-deficient mutant (E178A), rescued UV and camptothecin sensitivity in yeast lacking FEN-1.

Conclusions:

• FEN-1 possesses a novel GEN activity critical for resolving stalled replication forks.
• FEN-1 functions in conjunction with Werner syndrome protein and Replication protein A for efficient fork processing.
• FEN-1 plays a significant role in DNA damage tolerance and genomic stability in eukaryotic cells.