The eukaryotic cell cycle coordinates the accurate duplication and segregation of the genome during proliferation.  The large genomes of eukaryotic cells are replicated from multiple replication origins during S phase.  These origins are not activated synchronously at the beginning of S phase, but instead fire throughout S phase according to a pre-determined, cell type specific program.

Ensuring that each origin is efficiently activated once and only once during each S phase is crucial for maintaining the integrity of the genome.  This is achieved by a two-step mechanism.  The first step, known as licensing, involves the loading of the Mcm2-7 proteins into pre-replicative complexes (pre-RCs) at origins. In the second step, the MCM helicase is activated by a large set of ‘firing factors’. These two steps are differentially regulated by cyclin dependent kinase (CDK): licensing is inhibited by CDK, whilst firing requires CDK. As a consequence, licensing can only happen during G1 phase, when CDK activity is low, and origin firing cannot occur during G1 phase.

We have recently described the reconstitution of the initiation of eukaryotic DNA replication with purified proteins.  I will present recent results on the reconstitution of the entire replisome.   I will describe how chromatinised templates are replicated in vitro, how nucleosomes displaced during replication are re-deposited on nascent DNA, and how chromatin influences DNA replication origin choice and lagging strand synthesis.