Biochemical and Evolutionary Analysis of DNA Replication Initiation in Eukaryotic Cells

During the G1 phase of the cell division cycle licensing origins of DNA replication occurs and is followed by initiation of DNA replication from these origins in a defined and inherited temporal order during S phase. The vast majority of knowledge about the mechanism and control of licensing and subsequent replication has emerged from studies using the S. cerevisiae system, in which about 400 origins are licensed in G1 phase. In the origin licensing step, multiple DNA replication machinery components, specifically Orc1-6, Cdc6, Cdt1 and Mcm2-7, recognize origins of replication throughout the genome and form a pre-replication complex (pre-RC). ORC and Cdc6 recruit the Cdt1-Mcm2-7 complex to load the Mcm2-7 proteins onto the DNA, forming head-to-head MCM hexameric dimers that stay in an inactive state. Once activated by Cyclin-dependent (CDK) and Cdc7-Dbf4 (DDK) protein kinases and the recruitment of helicase activation factors, the helicase activities of the two MCM hexamers unwinds the DNA, initiating the replication process. In contrast to S. cerevisiae, it is estimated that there are approximately 50,000 origins in the human genome. The transition from licensing to initiation is tightly regulated both spatially and temporally. Understanding the features of an origin that determine its location in the genome, as well as the control and timing of origin activation remains an intriguing question in many eukaryotic cells. To address these issues, an evolutionary approach using biochemistry, structural biology and whole genome analyses has enabled comparison of origin location in multiple species. In S. cerevisiae, origins are located by the binding of ORC to specific DNA sequences. In contrast, in many yeasts and in human cells, indeed in most eukaryotes, origin location appears to be DNA sequence independent. Understanding the location of ORC binding sites in the genomes of multiple yeasts and in human cells and identification of proteins that interact with ORC and Cdc6 have advanced understanding of the mechanism of licensing of DNA replication in eukaryotes. Research was funded by a grant from the National Institute of General Medical Sciences, the Goldring FamilyFoundation and Cold Spring Harbor Laboratory.