Involvement of flap endonuclease 1 in the cleavage of stalled replication forks

Abstract

Flap endonuclease1 (Fen1) and Dna2 are two critical endonucleases that work conjointly for processing of Okazaki fragments in eukaryotes. Mus81-Mms4 is another structure-specific endonuclease to resolve stalled replication forks as well as toxic recombination intermediates. In this study, we found that Mus81-Mms4 suppresses the defect of dna2 mutations and that it has functional and physical interactions between Fen1. Mus81-Mms4 stimulated significantly Fen1 activity, accounting for its ability to restore the growth defect caused by dna2 mutation. Surprisingly, Fen1 dramatically stimulated the rate of Mus81-Mms4 cleavage of its all known substrates including regressed replication fork substrates. The ability of Fen1 to stimulate Mus81-Mms4 was independent of the catalytic function of Fen1. The mutual stimulation between Fen1 and Mus81-Mms4 occurred via a specific protein-protein interaction. Our $\It{in vitro}$ data indicate that Mus81-Mms4 and Fen1 are likely to act together during DNA replication via direct interaction. This is supported by the findings that $\It{fen1 mus81}$ or $\It{fen1 mms4}$ double mutants could not rescue of lethality. We discuss the significance of the physical interactions between Fen1, Dna2, and Mus81-Mms4 in context of DNA replication.