3 Assembly of Pre-replication Complexes

Abstract

DNA replication is tightly regulated such that each daughter cell receives exactly one copy of the parental cell’s DNA content upon cell division. Disturbing this controlled series of events can lead to genomic instability—a hallmark of cancer—and understanding the mechanism and regulation of DNA replication therefore becomes essential. Not surprisingly, the cell has developed intricate mechanisms and regulatory networks to guarantee the complete and accurate duplication of the genome. These mechanisms ensure that the DNA is replicated exactly once per cell cycle and that DNA replication is arrested in response to DNA damage and other replication stresses (e.g., low nucleotide levels). In many instances, the target of these regulatory mechanisms is a key intermediate in the initiation of DNA replication called the pre-replication complex (pre-RC).

The pre-RC is a multiprotein assembly that forms at all potential origins of replication. Originally identified on the basis of in vivo DNase I protection assays (Diffley et al. 1994), pre-RC formation requires at least four different proteins: the origin recognition complex (ORC), Cdc6, Cdt1, and the Mcm2-7 complex. Consistent with the central role of the pre-RC, the steps involved in its formation and the proteins involved are conserved across eukaryotic organisms (see Appendix, Table III). Formation of the pre-RC is initiated by the recognition of the origin DNA by ORC. ORC then recruits Cdc6, Cdt1, and the Mcm2-7 complex to the origin. After all of these proteins are present at the origin, a series of ATP-dependent events results in the loading of...