Reconstitution Of Regulated Eukaryotic Dna Replication With Purified Budding Yeast Proteins

Abstract

DNA replication is a fundamental process by which organisms copy their DNA. Biochemical reconstitution approaches in bacteria and viral systems have been instrumental in enhancing our mechanistic understanding of this process. However, a reconstituted system to study eukaryotic chromosomal DNA replication has been lacking. My thesis work focused on overcoming this deficiency by aiming to reconstitute eukaryotic DNA replication with purified budding yeast proteins, as chromosome replication is highly conserved from yeast to humans but best understood in budding yeast, Saccharomyces cerevisiae. Eukaryotic DNA replication initiates via a conserved two-step mechanism from multiple origin sites distributed along the length of each chromosome. In the first step, which is restricted to G1 phase, the core of the replicative helicase complex, Mcm2-7, is loaded in an inactive form at origins of replication, forming a pre-replicative complex (pre-RC). Subsequent activation of the Mcm2-7 helicase in the second step occurs exclusively in S-phase. Mcm2-7 loading has been previously reconstituted with purified proteins. However, the unusual double hexameric configuration of the Mcm2-7 complex bound around double stranded DNA observed in this reaction, while providing a molecular rationale for bi-directional origin activation, raised the question whether this structure is a true replication intermediate. Here, I show that reconstituted pre-RCs indeed support regulated replication of plasmid DNA in yeast cell extracts exhibiting the hallmarks of cellular replication initiation. Expanding on this observation, I have subsequently reconstituted DNA replication free in solution with purified budding yeast proteins in order to generate a system that allows for the biochemical study of all steps of eukaryotic DNA replication. The system recapitulates regulated bidirectional origin activation; synthesis of leading and lagging strands by the three replicative DNA polymerases Pol α, Pol δ and Pol ε and canonical maturation of Okazaki fragments into continuous daughter strands. A dual regulatory role for chromatin was uncovered during the replication of chromatinized templates: i) promoting origin dependence and ii) determining Okazaki fragment length by restricting Pol δ progression. Hence, this system provides a functional platform for the detailed mechanistic analysis of eukaryotic chromosome replication.