During S-phase of the cell cycle chromosomal DNA is usually replicated according to a complex replication timing program with megabase-sized domains replicating at different times. timing domains origin clusters and replication foci. We suggest that each timing domain name typically comprises several replicon clusters which are usually processed sequentially by replication factories. We discuss how replication might be regulated at different levels to create this complex business and the potential involvement of CDKs in this process. (frog) egg extracts.14 15 Nuclei prepared from CHO cells in mid G1 beyond the Timing Decision Point are programed to replicate according to the in vivo Rabbit polyclonal to Dcp1a. timing program.5 At 20 minutes the length of S-phase in the early Xenopus embryo is significantly shorter than that of the ～12 hour S-phase of CHO cells. When incubated in Xenopus egg extracts the CHO nuclei undergo a significantly accelerated timing program lasting ～2 hours.14 15 Replication labeling patterns resembling those observed in vivo develop sequentially during the in vitro incubation (Fig. 2). However in addition to the more discrete patterns discerned in vivo combined patterns (Types I/II II/III III/IV IV/V) were also observed. This suggests that in vitro later stages of the timing program can start while previous stages are still ongoing. Although most nuclei proceed to late replication patterns overall DNA replication is usually inefficient averaging less than 40% of template DNA replicated.14 15 Despite the ～6-fold reduction in S-phase length in vitro the amount of DNA synthesis associated with each replication pattern is proportionally similar to the time spent replicating each pattern in vivo.15 Taken together these results suggested that this replication program can progress to later timing stages before completing earlier steps. This means that replication initiation and the replication timing program are not tightly coordinated and so may be differentially regulated. Figure 2 Progression of CHO nuclei through the replication timing program in Xenopus egg extract. CHO nuclei incubated in Xenopus egg extracts were pulse labelled for 5 min at various occasions with Cy3-dUTP to directly visualize patterns of DNA replication. Shown … The Role of Cdks in S-Phase Progression During late mitosis and early G1 each replication origin is first loaded with Mcm2-7 to form a prereplicative complex TPCA-1 that licenses DNA for a single initiation event in the following S-phase (Fig. 1B).16 17 During S-phase each licensed origin is then activated by Cdks and Dbf4-dependent kinases (Ddks) to induce initiation. The combined action of these two kinases promotes the recruitment of replisome proteins to TPCA-1 the origin and the establishment of active replication forks. The number TPCA-1 of origins licensed in each cell cycle is in significant extra over the number of origins actually used during S-phase.18-20 Otherwise dormant origins can be activated when replication fork progression is inhibited providing a means to maintain DNA replication rates under conditions of replicative stress.21-23 In an unperturbed S-phase dormant origins are usually passively replicated and inactivated by forks initiated at an adjacent origin. The selection of origins for activation in the coming S-phase the ‘origin decision point’ occurs during G1 phase after the establishment of replication timing (Fig. 1B).24 25 It is unclear what makes some origins efficient and some origins inefficient (usually dormant). One possibility is usually that efficient origins are preferential targets for the initiating activities of Cdks and Ddks. We therefore TPCA-1 investigated the effect of modulating Cdk activity in the egg extract using the chemical Cdk inhibitor roscovitine and recombinant cyclin A.15 If strictly coupled both the rate of replication and progression through the timing program would respond to alteration of Cdk activity. Conversely if progression through the timing program was impartial of both then no effect on timing would be observed. Addition of increasing TPCA-1 concentrations of roscovitine to egg extract over a range of 1 1 to 100 μM reduced Cdk activity to near background levels. Consistent with previous reports the rate of DNA replication reduced with TPCA-1 comparable kinetics suggesting that Cdk activity is usually rate limiting for replication.26-29 Whereas roscovitine concentrations of 1-10 μM reduced the rate of DNA replication by up to ～50% the timing program was little affected. Only at roscovitine concentrations of 30 μM and.