Evidence for sequential and increasing activation of replication origins along replication timing gradients in the human genome.
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.
Institut de Biologie de l'Ecole Normale Supérieure (IBENS), CNRS UMR8197, Inserm U1024, Paris, France.
Guillaume
G
Guilbaud
Aurélien
A
Rappailles
Antoine
A
Baker
Chun-Long
CL
Chen
Alain
A
Arneodo
Arach
A
Goldar
Yves
Y
d'Aubenton-Carafa
Claude
C
Thermes
Benjamin
B
Audit
Olivier
O
Hyrien
D002469:Cell Separation; D019295:Computational Biology / Q000379:methods; D004261:DNA Replication; D005434:Flow Cytometry; D005821:Genetic Techniques; D015894:Genome, Human; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008957:Models, Genetic; D015233:Models, Statistical; D018741:Replication Origin; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017422:Sequence Analysis, DNA; D013997:Time Factors