Abstract
During cell division, the duplication of the genome starts at multiple positions called replication origins. Origin firing requires the interaction of rate-limiting factors with potential origins during the S(ynthesis)-phase of the cell cycle. Origins fire as synchronous clusters which is proposed to be regulated by the intra-S checkpoint. 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. 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.
Highlights
Eukaryotic genomes are duplicated in a limited time during the S phase of each cell cycle
Chk1 inhibition by UCN-01 did not result in the broader eye-toeye distribution predicted by random origin firing (Figure 2c,d), suggesting that other mechanisms than intra-S checkpoint are involved in the origin clustering
To decipher the regulation of origin firing by Chk1, we examined if the MM5 model could reproduce the replication program observed when the intra-S phase checkpoint was perturbed by the specific Chk1 inhibitor UCN-01 or by Chk1 over expression
Summary
Eukaryotic genomes are duplicated in a limited time during the S phase of each cell cycle. The mechanisms that regulate the timing of origin firing remain largely unknown [3,4,5,6,7,8]. 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 [9,10]. 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 [1]. MCM2-7 DHs that fail to fire are inactivated by forks emanating from neighboring fired origins [11].
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