Reconstitution of human DNA licensing and the structural and functional analysis of key intermediates

Abstract

SUMMARYHuman DNA licensing initiates the process of replication fork assembly. Specifically, this reaction leads to the loading of hMCM2-7 on DNA, which represents the core of the replicative helicase that unwinds DNA during S-phase. Here, we report the biochemical reconstitution of human DNA licensing using purified proteins, the structural and functional analysis of the process and reveal the impact of cancer-associated mutations on DNA licensing. We showed that thein vitroreaction is specific and results in the assembly of high-salt resistant hMCM2-7 double-hexamers, the final product of DNA licensing. We used ATPγS to block complex assembly at the hOrc1-5-Cdc6-Cdt1-MCM2-7 step. We observed that the assembly of this intermediate is independent of hOrc6, although hOrc6 enhances the loading of the second hMCM2-7 hexamer. The structural and mutational analysis of the hOrc1-5-Cdc6-Cdt1-MCM2-7 complex provides insights into hORC-Cdc6 dependent recruitment of hMCM2-7 via five hMcm winged-helix domains. The structure highlights how hOrc1 activates the hCdc6 ATPase, while the analysis of hOrc1 and hCdc6 ATPase mutants uncovered an unexpected role for hCdc6 ATPase in complex disassembly. The structure highlights that Cdc6 binding to Orc1-5 stabilises Orc2-DNA interactions and supports Mcm3-dependent recruitment of MCM2-7. Finally, the structure allowed us to locate cancer-associated mutations at the hCdc6-Mcm3 interface, which showed specific helicase loading defects.