Illuminating the molecular pathways of DNA replication initiation.

Eukaryotic DNA replication occurs only once per cell cycle to maintain genome stability and avoid the onset of cancer. To achieve this, eukaryotes have evolved mechanisms controlling the timing of replication origin activation. In G1, DNA-loading of the MCM replicative helicase (inactive form) licenses origins. Activation occurs in S phase, when Cdc45, GINS and Pol epsilon engage MCM to form the CMGE holohelicase that unwinds DNA for replication. We previously provided the first structural snapshots of origin activation using biochemical reconstitution with purified yeast proteins. We will now assemble a molecular movie of the whole process, including MCM loading, DNA opening and priming. Higher eukaryotes use a mix of conserved mechanisms and new pathways to initiate replication. To understand the molecular basis, we will characterise replicating chromatin established with Xenopus egg extracts. Using this system, we will employ cryo-electron microscopy/biochemistry to study purified origin-activation complexes or cryo-electron tomography of replicating nuclei to understand initiation in the native context. Resulting information on the cascade of molecular events and the factors essential for replication will be used in biochemical reconstitution efforts to describe the structural mechanism of human origin activation. Collectively, our results will elucidate core mechanisms of replication initiation across eukaryotes.