i new pre-RC formation inhibited existing pre-RC activation
The tight connection between pre-RC function. Cdk levels, and die cell cycle ensures that the eukaryotic genome is replicated only once per cell cycle (Figure 8-32). Active Cdk is absent during CI, whereas elevated levels of Cdk are present during the remainder of the cell cycle
FIGURE 8-32 Cell cycle regulation of Cdk activity and pre-RC formation. In G1,
Cdk levels are low and new pre-RC complexes can form but cannot be activated. Dunng S phase, the elevated levels of Cdk activity trigger the initiation of DNA replication and prevent any new pre-RC complex formation on newly replicated DNA. Once a pre-RC is used for the initiation of replication, it is necessarily dismantled (recall that at least one key component of the pre-RC, tho Mem compfcx, becomes part of the replication fort;). Similarly, replication of pre-RC associated DNA also causes destiuction of the complex (not shown). Because Cdk levels remain high until the end of mitosis, no new pre-RC complexes can be formed until chromosome segregation is complete. Without new pre RC complexes, re-initiation is impossible.
activity phase Cdk levels high pre-RC activation no pre-RC activation assembly phase Cdk levels low
(S, C2, and M phases). Thus, during each eel) cycle there is only one opportunity for pre-RCs to form (during Gl) and only one opportunity far those pre-RCs to he activated (during S, G2, and M—although in practice all pre-RCs are activated or disrupted by replication forks in S phase).
Pre-RCs are disassembled after they are activated or after the DMA to which they are bound is replicatcd- These exposed replicators are then available for new pre-RC formation and rapidly bind to ORC, Despite the presence of the initiator at these sites, the elevated levels of Cdk activity in S, G2, and M physe cells prevents the association erf the other members of the pre-RC complex with ORC. It is only when cells segregate their chromosomes and complete cell division that Cdk activity is eliminated and new pre-RC complexes can form.
Similarities between Eukaryotic and Frokaryotic DNA Replication Initiation
Now that we have described initiation in eukaryotes and prokaryotes, it is clear that the general principles of replication initiation are the same in both cases. The first step is the recognition of the replicator by the initiator protein. The initiator protein in combination with one or more helicase loading proteins, recruit the DNA helicase to the replicator. The helicase (and potentially other proteins at the origin in eukaryotes) generate a region of ssDNA that can act as a template for RNA primer synthesis. Once primers are synthesized, the remaining components of the replisome assemble through interactions with the resulting primer:template junction.
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