Post-translational Modifications by Acyl Groups Regulate FEN1's Activities and Play Essential Roles in Cell Proliferation and DNA Repair.

Abstract

Flap endonuclease 1 (FEN1), well known for its structural-specific nuclease, possessing 5'-flap endonuclease and 5'-3' exonuclease ac-tivities, is mainly involved in DNA replication and repair. Protein lysine acetylation is an important posttranslational modification that could regulate numerous proteins' activity, subcellular localization, protein-protein interac-tion etc., and influences many biological processes. Our previous studies on integrated succinylome profiles found that succinylation and acetylation lev-els of FEN1 would change under different conditions. Succinylation at FEN1 Lys200 site results in the accumulation of damaged DNA and in-creased susceptibility to fork-stalling agents. The interplay with other forms of modification could affects its protein interaction affinity and thus con-tribute to genome stability. This article studied the biological role of FEN1 by acyl modification in HeLa cells. In order to explore the function of FEN1 acylation in cells, we mimicked the presence or ab-sence of acetylation or succinylation by mutating key amino acids to glutam-ic acid and glutamine. We carried out a series of experiments including cell cycle, MTS, enzyme kinetics measurements, immunofluorescence and so on. The absence of acylation of FEN1 leads to the blocked cell cycle process and the reduced efficiency of FEN1 on its DNA substrates, affect-ing the interaction of FEN1 with both repair and replication related proteins and thus its role in the repair of DNA damage. We have veri-fied acyl groups could modify Lys125, Lys252 and Lys254 of FEN1. Acyl-ation level of these three is important for enzyme activity, cell proliferation and DNA damage response, thus contributing to genome stability.