Evidence for Sequential and Increasing Activation of Replication Origins along Replication Timing Gradients in the Human Genome

Abstract

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 sorted into four temporal compartments of S phase shows that replication origins are spaced at 40 kb intervals and fire as small clusters whose synchrony increases during S phase and that replication fork velocity (mean 0.7 kb/min, maximum 2.0 kb/min) remains constant and narrowly distributed through S phase. However, multi-scale analysis of a genome-wide replication timing profile shows a broad distribution of replication timing gradients with practically no regions larger than 100 kb replicating at less than 2 kb/min. Therefore, HeLa cells lack large regions of unidirectional fork progression. Temporal transition regions are replicated by sequential activation of origins at a rate that increases during S phase and replication timing gradients are set by the delay and the spacing between successive origin firings rather than by the velocity of single forks. Activation of internal origins in a specific temporal transition region is directly demonstrated by DNA combing of the IGH locus in HeLa cells. Analysis of published origin maps in HeLa cells and published replication timing and DNA combing data in several other cell types corroborate these findings, with the interesting exception of embryonic stem cells where regions of unidirectional fork progression seem more abundant. These results can be explained if origins fire independently of each other but under the control of long-range chromatin structure, or if replication forks progressing from early origins stimulate initiation in nearby unreplicated DNA. These findings shed a new light on the replication timing program of mammalian genomes and provide a general model for their replication kinetics.

Genomic Analysis of the DNA Replication Timing Program during Mitotic S Phase in Maize (Zea mays) Root Tips

“…The presence of some relatively sharp peaks in mid S suggests these may be initiation zones ( Figure 8B). However, mid peaks are generally lower intensity with gentler slopes than the peaks found in early S, consistent with the idea that high efficiency origins fire in early S followed by a "cascade" of lower efficiency origins in mid S (Guilbaud et al, 2011). The generally lower intensity mid S pattern is also consistent with the possibility that many mid S loci are passively replicated by elongation from earlier initiation events ( Figure 8C).…”

“…Most studies in mammalian cells agree that much of the replication occurs in large coordinated domains (0.2-2 Mb) that presumably contain multiple origins (Farkash-Amar et al, 2008;Guilbaud et al, 2011;Hyrien, 2016;Klein and Gilbert, 2016). The size of replication domains in mammals is further supported by results from chromatin capture experiments that have defined topologically associated domains.…”

“…Some have hypothesized that these TTRs consist of unidirectional replication forks spreading from origins active earlier in S phase (Hiratani et al, 2008;Ryba et al, 2010), while others have argued that such regions contain origins activated in a "cascading" or "domino" pattern by earlier firing origins nearby (Guilbaud et al, 2011). There has been much less debate over the patterns of activation of origins firing within the constant timing region domains, as these are thought to activate roughly synchronously in clusters (Jackson and Pombo, 1998;Blow et al, 2011;Boulos et al, 2015;Klein and Gilbert, 2016).…”

The Involvement of Acidic Nucleoplasmic DNA-binding Protein (And-1) in the Regulation of Prereplicative Complex (pre-RC) Assembly in Human Cells

“…Fork density in cells treated with siGl2 was 3.19 forks/megabase (Fig. 6D), similar to a previous report by Guilbaud et al (43), whereas the fork density in cells depleted of And-1 by either siAnd-1-1 or siAnd-1-2 was 2.61 forks/megabase and 2.78 forks/megabase, respectively (Fig. 6D).…”

Activation of new replication foci under conditions of replication stress

“…The existing knowledge about activation of replication origins under conditions of stress is derived primarily from studies of DNA fibers pulled out of nuclei. [7][8][9][10][11][12] We note, however, that these studies do not provide information about distances between the originally active and newly activated replication regions in 3D space of individual nuclei. They also provide no information about replication rates in individual replication factories, nor about the spectrum of reactions of individual cells within a population to stress.…”