Cell Cycle Control of Chromosomal Replication
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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 every cell cycle (Ref.1). 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 down regulated in response to agents that damage DNA or that block DNA replication.

The initiation of chromosomal DNA replication in eukaryotic cells is a highly regulated process and requires a series of complex events including 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 (Ref.2). ORC (Origin Recognition Complexes), which are associated specifically with replication origin throughout the cell cycle serve as 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 preRC (pre Replication Complex) at origins of replication, before the initiation of DNA synthesis. In the absence of CDC6, cells fail to initiate DNA replication and undergo a “reductional” mitosis, in which the unreplicated chromosomes are randomly segregated to the spindle poles. In addition to CDC6, ORC also binds to CDKs (Cyclin-Dependent Kinases) (Ref.3). This association is followed by the loading of MCM (Minichromosome Maintenance) protein complexes onto the replication origins, leading to the formation of preRCs. CDT1 is also required for MCM binding to chromatin. After the MCM loading, S-phase CDKs phosphorylates CDC6/CDC18, and this phosphorylation leads to the rapid degradation of CDC6/CDC18 to prevent reinitiation (Ref.4). 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 (Replication Protein-A, single-stranded DNA-binding protein) and DNA Polymerases, are loaded onto RCs (Ref.5). After DNA synthesis is initiated, some of the MCM components may leave the complexes, resulting in postRC.

Depletion of CDC6 prevents replication initiation and results in a reductional mitosis in which cells randomly segregate their unreplicated chromosomes. Under these circumstances, the G1-M checkpoint is not operating. These 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 (Ref.6).