Repair complexes of FEN1 endonuclease, DNA, and Rad9-Hus1-Rad1 are distinguished from their PCNA counterparts by functionally important stability

摘要

Processivity clamps such as proliferating cell nuclear antigen (PCNA) and the checkpoint sliding clamp Rad9/Rad1/Hus1 (9-1-1) act as versatile scaffolds in the coordinated recruitment of proteins involved in DNA replication, cell-cycle control, and DNA repair. Association and handoff of DNA-editing enzymes, such as flap endonuclease 1 (FEN1), with sliding clamps are key processes in biology, which are incompletely understood from a mechanistic point of view. We have used an integrative computational and experimental approach to define the assemblies of FEN1 with double-flap DNA substrates and either proliferating cell nuclear antigen or the checkpoint sliding clamp 9-1-1. Fully atomistic models of these two ternary complexes were developed and refined through extensive molecular dynamics simulations to expose their conformational dynamics. Clustering analysis revealed the most dominant conformations accessible to the complexes. The cluster centroids were subsequently used in conjunction with single-particle electron microscopy data to obtain a 3D EM reconstruction of the human 9-1-1/FEN1/DNA assembly at 18-A resolution. Comparing the structures of the complexes revealed key differences in the orientation and interactions of FEN1 and double-flap DNA with the two clamps that are consistent with their respective functions in providing inherent flexibility for lagging strand DNA replication or inherent stability for DNA repair.