Structure and function studies of replication initiation factors

Abstract

We have used negative stain EM and cryo-EM to visualize step by step the replication initiation events in S. cerevisiae, as the process is driven forward by the interplay of a dozen or so macromolecular initiation factors, leading to the establishment of pre-replication complexes (pre-RC) at each origin of DNA replication. This work took advantage of our ability to reconstitute the Mcm2-7 loading reaction with purified proteins. We determined the architecture of several previously known replication initiation complexes such as ORC, ORC-Cdc6 on DNA, and the Mcm2-7 double hexamer. We also captured by EM reaction intermediates such as the ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) and the ORC-Cdc6-Mcm2-7-Mcm2-7 (OCMM) that had evaded previous biochemical identification. In this chapter, we describe what we have learnt about the structure and interaction with origin DNA of the replication initiators. We further discuss what may be expected in the coming years as cryo-EM is becoming a near-atomic resolution structural tool, thanks to the recent advent of the direct electron detector.