We showed that even during an unperturbed S phase in Xenopus egg extracts, Chk1 inhibits origin firing away from but not near active forks. Our first model for DNA replication in Xenopus egg extracts (which combined time-dependent changes in the availability of a limiting replication factor, and a fork-density dependent affinity of this factor for potential origins) was used to model the regulation of DNA replication by the intra-S checkpoint. Experiments using Xenopus egg extracts suggested that the checkpoint mainly adjusts the rate of DNA synthesis by staggering the firing time of origin clusters. Experiments in human cells under low replication stress conditions showed that Chk1 inhibits the activation of new replication factories while allowing origin firing to continue within active factories. The metazoan functional analogue of Rad53 is Chk1. DNA replication stress, through the activation of the S-phase checkpoint kinase Rad53, can inhibit origin firing by phosphorylating and inhibiting Sld3 and Dbf4. It is now well accepted that the intra-S phase checkpoint regulates origin firing during both unperturbed and artificially perturbed S phase. Modulating origin firing propensity by the probability to form loops between forks and nearby potential origins resulted in a better fit to the data without affecting I t. Simulations incorporating this extracted I t reproduced the mean eye length, gap length and eye-to-eye distance, but the experimental eye-to-eye distance distribution appeared “peakier” than the simulated one. The extracted I t markedly increased during S phase. This model allowed the extraction of a time-dependent rate of replication initiation, I t, from the measured eye lengths, gap lengths and eye-to-eye distances on combed DNA molecules ( Figure 1a). named “mean-field hypothesis”), (ii) that firing of origins are independent events and (iii) that fork speed is constant was proposed. Among these six factors, Cdc45 is the only one to travel with the replication fork.Ī mathematical model based on the assumptions (i) that the probability of firing of each replication origin can be replaced by the averaged probability of firing calculated over all degree of freedom of origin firing process (MCM2-7 DH density, genomic position, chromatin compaction, nucleosome density, etc. The six initiation factors Sld2 (RecQ4 in Xenopus), Sld3 (Tresline in Xenopus), Dpb11 (TopBP1 in Xenopus), Dbf4 (Drf1 in Xenopus), Sld7 (MTBP in Xenopus) and Cdc45 are expressed at concentrations significantly lower than the MCM complex and core replisome components, suggesting that they may be rate-limiting for origin firing. Origin firing requires S-phase cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK) activities as well as the CDK targets Sld2 and Sld3 and the replisome-maturation scaffolds Dpb11 and Sld7 in S. MCM2-7 DHs that fail to fire are inactivated by forks emanating from neighboring fired origins. ![]() During S phase, only a fraction of the MCM2-7 DHs are activated to form a pair of active Cdc45-MCM2-7-GINS (CMG) helicases and establish bidirectional replisomes. The core motor component of the replicative helicase, the MCM2-7 complex, is loaded on chromatin from late mitosis until the end of G1 phase as an inactive head-to-head double hexamer (DH) to form a large excess of potential origins. This model implies that the observed origin clustering emerges from the apparent synchrony of origin firing in regions with high probability of origin firing and challenge the assumption that the intra-S checkpoint is the main regulator of origin clustering. By modelling the unchallenged, the checkpoint-inhibited and the checkpoint protein Chk1 over-expressed replication pattern of single DNA molecules from Xenopus sperm chromatin replicated in egg extracts, we demonstrate that the quantitative modelling of data requires: (1) a segmentation of the genome into regions of low and high probability of origin firing (2) that regions with high probability of origin firing escape intra-S checkpoint regulation and (3) the variability of the rate of DNA synthesis close to replication forks is a necessary ingredient that should be taken in to account in order to describe the dynamic of replication origin firing. Origins fire as synchronous clusters which is proposed to be regulated by the intra-S checkpoint. Origin firing requires the interaction of rate-limiting factors with potential origins during the S(ynthesis)-phase of the cell cycle. During cell division, the duplication of the genome starts at multiple positions called replication origins.
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