Replication fork movement sets chromatin loop size and origin choice in mammalian cells

Abstract

Genome stability requires one, and only one, DNA duplication at each S phase. The mechanisms preventing origin firing on newly replicated DNA are well documented, but much less is known about the mechanisms controlling the spacing of initiation events(2,3), namely the completion of DNA replication. Here we show that origin use in Chinese hamster cells depends on both the movement of the replication forks and the organization of chromatin loops. We found that slowing the replication speed triggers the recruitment of latent origins within minutes, allowing the completion of S phase in a timely fashion. When slowly replicating cells are shifted to conditions of fast fork progression, although the decrease in the overall number of active origins occurs within 2 h, the cells still have to go through a complete cell cycle before the efficiency specific to each origin is restored. We observed a strict correlation between replication speed during a given S phase and the size of chromatin loops in the next G1 phase. Furthermore, we found that origins located at or near sites of anchorage of chromatin loops in G1 are activated preferentially in the following S phase. These data suggest a mechanism of origin programming in which replication speed determines the spacing of anchorage regions of chromatin loops, that, in turn, controls the choice of initiation sites.

DNA replication is altered in Immunodeficiency Centromeric instability Facial anomalies (ICF) cells carrying DNMT3B mutations

“…Interestingly, fork speed was significantly higher and S-phase length was shorter in two ICF cell lines out of three. Changes in replication fork speed may be associated with changes of inter-origin distances; 22,23 however, this did not occur in ICF cell lines that had inter-origin distances comparable to controls. A DNA replication defect generalized to the whole genome was unexpected considering that major epigenetic changes in ICF patients occur in heterochromatic regions (reviewed by Ehrlich et al 26 ) and that the shift of replication timing is restricted to heterochromatic regions.…”

“…Changes in replication fork speed may be associated with compensatory changes of inter-origin distances. 22,23 To check whether the increase in fork speed in ICF3 and ICF4 was associated with a change of the inter-origin distance, we measured the center-to-center distance between two adjacent CldU tracks (inter-track distance). This measurement is currently used to estimate inter-origin distance.…”

Hydroxyurea induces de novo copy number variants in human cells

“…This result could be due to selection pressures inherent to generating fibroblast clones harboring CNVs in these regions or, alternatively, a more complex relationship between fragile site instability and CNVs secondary to their common induction by replication stress. Both depletion of nucleotide pools by HU and polymerase inhibition by APH lead to reduced replication fork rate, fork stalling, and activation of dormant origins (51)(52)(53)(54). It has recently been shown that dormant origins are only activated in early replicating regions, whereas origin firing is suppressed by S-phase checkpoints in late-replicating regions of the genome (54).…”

Chk1–cyclin A/Cdk1 axis regulates origin firing programs in mammals

“…Molecular combing of single DNA molecules demonstrated that Chk1 depletion resulted in a clear reduction in origin spacing (∼40% of control cells) as well as in the rate of fork elongation throughout the labeling period. However, since slowing the replication speed very rapidly triggers the recruitment of latent origins (Courbet et al 2008), it is not clear at this stage whether Chk1 regulates either or both activation of origins or fork velocity. Molecular combing also revealed that loss of Chk1 frequently stalls and collapses active forks.…”