Defining the Superfamily Conserved Mechanism for Flap Endonucleases FEN1 and XPG Specificity for 5' Flap DNA and DNA Bubbles, Respectively, by Hybrids Methods of Crystallography, SAXS, EM, and Computation

Abstract

Flap endonuclease 1 (FEN1) and XPG are essential 5′ nuclease superfamily endonucleases in DNA replication and repair. FEN1 incises in the dsDNA region adjacent to 5′ flaps, while XPG incises in the dsDNA region adjacent to DNA bubbles. We have used a hybrids method analysis combining crystallography, Small Angle X-ray Scattering (SAXS), Electron Microscopy (EM), and computation to characterize FEN1 specificity and activity on double flap substrates, and in the presence of sliding clamps 9-1-1 and PCNA and XPG specificity for DNA bubbles. Our crystallographic work shows a 5′nuclease superfamily conserved mechanism for resolving aberrant DNA structures that involves both structural motifs and flexibility. Our structures of product and substrate-bound complexes suggested FEN1 resolves 5′ flaps using a dsDNA binding - ssDNA incision mechanism. The structure revealed FEN1 binding to a bent duplex DNA structure and an active site shielded by a helical gateway that would select for single-stranded DNA or RNA to reach the active site. Key structural elements mediating duplex DNA binding, substrate specificity and activity are superfamily conserved in our XPG catalytic domain crystal structure. Further computational and EM work with FEN1 complexes to sliding clamps 9-1-1 and PCNA showed distinct functionally-relevant differences in how FEN1 interacts with the sliding clamps and how the sliding clamps interact with upstream duplex DNA. We propose that the PCNA complex is more dynamic, consistent with PCNA's role in replication and that the 9-1-1 complex is more stable, consistent with 9-1-1 acting locally at the DNA damage.