Study Of Structure-specific Endo-Nuclease FEN1Mutants And Their Roles In Carcinogenesis

1. Programmed PTMs mediate FEN1degradation and genome stabilityFEN1, as a structure-specific endo-and exo-nuclease, plays a critical role in genome stability through multiple DNA metabolic pathways, including DNA replication, repair, and apoptotic DNA fragmentation. Persistent presence of the nuclease in various cell cycle phases is predicted to have deleterious effects on the cell cycle and genome stability. Here, we report that degradation of FEN1nuclease is cell cycle dependent and precisely programmed by sequential post-translational modifications. Specifically, phosphorylated FEN1falls off the DNA replication complex at late S-phase, and is the species that is degraded. Phosphorylation is a signal for SUMOylation, which subsequently induces ubiquitylation-mediated proteasome degradation. Disruption of this program causes cellular resistance to FEN1degradation, stabilization of cyclins B and, significant delay in phases Gl and G2/M of the cell cycle, and aberrant chromosomal segregation. Herein, we demonstrate a novel mechanism that controls the balance of nuclease activities in cells.2. High Risk of Benzo[a]pyrene-induced Lung Cancer in E160D FEN1Mutant MiceFlap endonuclease1(FEN1), a member of the Rad2nuclease family, possesses5'flap endonuclease (FEN),5'exonuclease (EXO), and gap-endonuclease (GEN) activities. The multiple, structure-specific nuclease activities of FEN1allow it to process different intermediate DNA structures that result from numerous DNA replication and repair pathways. We previously identified a group of FEN1mutations and single nucleotide polymorphisms that impair FEN1's EXO and GEN activities in human cancer patients. In previous studies, we also established a mouse model carrying the E160D FEN1mutation, which models the mutations seen in humans, and observed that FEN1mutant mice spontaneously develop lung cancer in response to base damaging agents. An important unanswered question is whether individuals carrying such FEN1mutations are more susceptible to tobacco smoke and have an earlier onset of lung cancer. Here, we report our studies on E160D mutant mice exposed to benzo[α]pyrene (B[a]P), a major DNA damaging compound found in tobacco smoke. We demonstrate that FEN1employs its GEN activity to cleave model bubble substrates resembling the intermediate DNA structures that arise during nucleotide excision repair. Mice carrying the E160D mutation, which is deficient in GEN activity, had defects in the repair of B[α]P adducts. As a consequence, E160D cells accumulated more DNA strand breaks, chromosomal breakage, and chromosome instabilities in response to B[a]P exposure. Furthermore, E160D mice had a higher frequency of lung adenocarcinoma in response to exposure to B[a]P than did wild-type mice. Thus, our current study suggests that individuals carrying GEN-deficient FEN1mutations are predisposed to B[a]P-induced cancer development.