Cell Cycle Control of Chromosomal Replication

The stable propagation of genetic information requires that the entire genome of an organism be faithfully replicated only once in each cell cycle. In eukaryotes, this replication is initiated at hundreds to thousands of replication origins distributed over the genome, each of which must be prohibited from re-initiating DNA replication within a cell cycle. Initiation of DNA replication occurs by a two step process. In the first step, initiation proteins are assembled onto the replication origin in a step-wise fashion to yield an initiation complex. In the second step, the initiation complex is activated by protein kinases, resulting in the establishment of replication forks. This process is tightly regulated such that initiation at a given replication origin occurs only once per cell cycle. In addition, initiation is downregulated in response to agents that damage DNA or block DNA replication.Chromosomal DNA replication in eukaryotic cells requires a series of complex events which include recognition of origins, firing of replication origins, loading of DNA polymerases onto origins, and elongation of newly synthesized DNA. Initiation of DNA replication takes place only at specific loci on the chromosomal DNA termed replication origins. origin recognition complex (ORC), which is associated specifically with replication origin throughout the cell cycle, serves as a hallmark of the origins and is highly conserved. Newly synthesized CDC6/CDC18 temporally associates with ORC at the G1-S boundary and is essential for assembly of pre replication complex (preRC) at origins of replication before the initiation of DNA synthesis. This is followed by the loading of minichromosome maintenance (MCM) protein complex onto the replication origins leading to the formation of preRCs. CDT1 is also required for MCM binding to chromatin. After MCM loading, S-phase CDK phosphorylates CDC6/CDC18, and this phosphorylation leads to the rapid degradation of CDC6/CDC18 to prevent re-initiation. Preceding the firing of origins, CDC7-Dbf4 kinase complexes phosphorylate MCM proteins in preRCs and CDC45 is loaded onto preRCs at the replication origins. Finally, replication proteins, such as RPA and DNA Polymerases, are loaded onto RCs.Depletion of CDC6 prevents replication initiation and results in a reductional mitosis in which cells randomly segregate their unreplicated chromosomes. Checkpoints act to inhibit subsequent cell cycle events if there is a delay or error in a preceding stage. For instance, the S-phase checkpoint acts to inhibit the metaphase to anaphase transition when DNA damage or incomplete DNA replication occurs during S phase.