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Although replication proteins are conserved among eukaryotes, the sequence requirements for replication initiation differ between species. In all species, however, replication origins fire asynchronously throughout S phase. The temporal program of origin firing is reproducible in cell populations but largely probabilistic at the single-cell level. The(More)
BACKGROUND Eukaryotic cells seem unable to monitor replication completion during normal S phase, yet must ensure a reliable replication completion time. This is an acute problem in early Xenopus embryos since DNA replication origins are located and activated stochastically, leading to the random completion problem. DNA combing, kinetic modelling and other(More)
Genome-wide replication timing studies have suggested that mammalian chromosomes consist of megabase-scale domains of coordinated origin firing separated by large originless transition regions. Here, we report a quantitative genome-wide analysis of DNA replication kinetics in several human cell types that contradicts this view. DNA combing in HeLa cells(More)
Initiation of DNA replication is tightly controlled during the cell cycle to maintain genome integrity. In order to directly study this control we have previously established a cell-free system from human cells that initiates semi-conservative DNA replication. Template nuclei are isolated from cells synchronized in late G1 phase by mimosine. We have now(More)
In higher eukaryotes, replication program specification in different cell types remains to be fully understood. We show for seven human cell lines that about half of the genome is divided in domains that display a characteristic U-shaped replication timing profile with early initiation zones at borders and late replication at centers. Significant overlap is(More)
Replication origins in Xenopus egg extracts are located at apparently random sequences but are activated in clusters that fire at different times during S phase under the control of ATR/ATM kinases. We investigated whether chromosomal domains and single sequences replicate at distinct times during S phase in egg extracts. Replication foci were found to(More)
In this protocol, we describe the use of the LastWave open-source signal-processing command language (http://perso.ens-lyon.fr/benjamin.audit/LastWave/) for analyzing cellular DNA replication timing profiles. LastWave makes use of a multiscale, wavelet-based signal-processing algorithm that is based on a rigorous theoretical analysis linking timing profiles(More)
Sperm chromatin incubated in Xenopus egg extracts undergoes origin licensing and nuclear assembly before DNA replication. We found that depletion of DNA topoisomerase IIα (topo IIα), the sole topo II isozyme of eggs and its inhibition by ICRF-193, which clamps topo IIα around DNA have opposite effects on these processes. ICRF-193 slowed down replication(More)
Replication of mammalian genomes starts at sites termed replication origins, which historically have been difficult to locate as a result of large genome sizes, limited power of genetic identification schemes, and rareness and fragility of initiation intermediates. However, origins are now mapped by the thousands using microarrays and sequencing techniques.(More)
Despite intense investigation, human replication origins and termini remain elusive. Existing data have shown strong discrepancies. Here we sequenced highly purified Okazaki fragments from two cell types and, for the first time, quantitated replication fork directionality and delineated initiation and termination zones genome-wide. Replication initiates(More)