Investigating the DNA replication machinery composition and function using proteomic and chemical-biology approaches

Abstract

The process of DNA replication is essential for cellular life, but must be highly regulated to ensure that damage to the genome or irregularity in copy number does not ensue. In particular, the mechanism behind the loading and activation of the eukaryotic replicative helicase, MCM2-7, onto DNA has been investigated extensively. However, despite progress afforded by the advent of cryo-EM to reveal the structures of multi-subunit protein complexes, the molecular mechanism underlying the activation of the replicative helicase has yet to be fully elucidated. In particular, the precise temporal order of initiation factor recruitment and release, and how they affect complex progression, is not known. Additionally, many in vivo specific factors such as chromatin factors, accessory helicases and regulatory pathways are not well understood. This research set out to establish a more complete description of the interaction partners of the MCM2-7 helicase in vivo prior to, during, and after replicative helicase activation, by applying a combination of chemical-biology and proteomic techniques. Small molecule-inducible protein linkages were introduced into the MCM2-7 complex, to arrest the complex at different stages of helicase activation. This enabled investigation of transient conformational states in vivo. A versatile proteomic method utilising in vivo cross-linking and affinity enrichment of MCM2- 7 helicase interactomes followed by LC-MS/MS analysis was established, termed ChIP-MS. This technique enabled characterisation of the composition of replication complexes throughout the cell cycle, as well as in the context of the arrest states generated by the linkage constructs. Stabilisation of the in vivo complexes by cross-linking allowed the detection and compositional analysis of distinct sub-populations of replication complexes over multiple cell cycle stages and identified several factors and pathways, such as chromatin remodellers and accessory helicases, that may have a previously understudied or novel role in the replication process.