교차접합 DNA 치유를 위한 FAN1 제한효소의 구조적 생화학적 연구

Abstract

DNA Interstrand cross-link (ICL) is highly cytotoxic DNA lesion that covalently crosslinks 2 bases at opposite strand. ICL leads to replication fork stalling and transcriptional failure, therefore highly cytotoxic. Fancd2/FancI-associated nuclease1 (FAN1) is 5’ flap DNA structure specific endonuclease/exonuclease which recognizes 5’ flap DNA, removes 5’ flap by cleaving 4nt upstream of flap, followed by 5’-3’ exonucleolytic cleavage. together with several biochemical and cellular experiment, FAN1 has proposed as ICL unhooking enzyme along with XPF/ERCC1, SLX1/SLX4 and MUS81/EME1, but ICL unhooking mechanism and rescue of chromosome abnormality of FAN1 in eukaryotic cells is still enigmatic. In this thesis, I would present structure of bacterial FAN1 homologue, Pseudomonas aeruginosa FAN1 (PaFAN1) bound to 5’ flap DNAs with ssDNA gap which mimics initial product by endonucleolytic activity of PaFAN1. The Structures shows later stage of nuclease activity of PaFAN1, which may represent completion of ICL unhooking together with previously reported FAN1 structures bound to 5’ flap DNAs. The structures revealed novel structural feature ‘basic patch’ and following biochemical experiments further demonstrated that this patch is important especially for DNA exonuclease activity inducing conformational change in substrate DNA for subsequent cleavage. Biochemical experiments showed that both PaFAN1 and HsFAN1ΔUBZ were able to unhook the lesion by incising both sides of ICL through combinatorial action of endo/exonuclease and mutation on the basic patch reduced nuclease activity as well as ICL unhooking process. This is the first and only structure that showing the mechanism of endonucleolytic activity followed by exonucleolytic activity. I suggest unique and conserved ICL unhooking mechanism of FAN1 supported by structural and biochemical evidences and also how bacterial FAN1 evolved and related with eukaryotic FAN1.