![]() Zones of predominant initiation (termination) can then be detected as upward (downward) slopes in replication fork directionality (RFD= R−L) profiles ( Fig. The principle is to isolate and sequence Okazaki fragments to reveal the proportions of rightward- ( R) and leftward- ( L) moving forks throughout the genome. The low resolution of skew and replication timing profiles precluded precise location of initiation and termination events along N/U-domains.Ī novel method that permits a high-resolution, quantitative analysis of replication fork initiation, progression and termination has been recently developed in Saccharomyces cerevisiae 27, 28. Furthermore, N/U-domains are much longer (0.5–3 Mb) than typical inter-origin distances (100–200 kb) suggesting that additional intradomain origins may fire in a sequential pattern from N/U-domain borders to centres 15, 25, 26. However, detectable N/U-domains together cover only one-third to one-half of the human genome and it is unclear if replication of the remainder follows similar rules. When compared with somatic cell replication timing profiles 20, 21, 22, skew N-domains indeed coincided with U-shaped domains of replication timing, with borders (skew jumps) replicating early and centres replicating later 15, 21, 23, 24, 25. Between jumps, the skew decreased in a linear manner, suggesting a progressive inversion of the average direction of replication (in germline cells) across N-shaped domains of skew, perhaps due to the random termination between the two origins 19. ![]() Upward skew jumps similar to bacterial origins have been detected at 1,546 sites in the human genome 17, 18. Thus, detection of an abrupt sign inversion of the skew is a standard tool to predict origin and termini in bacteria 16. In most bacterial genomes, the GC and TA skews S GC=( G−C)/( G+C) and S TA=( T−A)/( T+A) are strictly correlated with replication direction, suggesting that mutational asymmetry of the leading and lagging strands led to skew accumulation over evolutionary times 14. In addition, these methods cannot precisely quantify origin efficiency nor map termination.Īn orthogonal method to map replication is based on the analysis of nucleotide compositional asymmetries of the two DNA strands 1, 14, 15. 9) putative origins detected genome-wide). The number and position of origins thus detected present discrepancies 1, 3 (from 12,000 (ref. Genome-wide identification of mammalian replication origins 1, 2, 3 has been attempted by various methods including trapping of replication-bubbles 4, 5, purification of RNA-primed single DNA strands 6, 7, 8, 9, 10, 11, immunoprecipitation of BrdU-labelled DNA 12 or chromatin immunoprecipitation of origin recognition complex (ORC) 13, followed by microarray hybridization or high-throughput sequencing. This novel panorama of replication reveals how chromatin and transcription modulate the initiation process to create cell-type-specific replication programs. Initiation zones are enriched in origin recognition complex (ORC)-binding sites and better align to origins previously mapped using bubble-trap than λ-exonuclease. Initiation zones are enriched in open chromatin and enhancer marks, even when not flanked by genes, and often border ‘topologically associating domains’ (TADs). Initiation and termination zones are frequently contiguous, sometimes separated by regions of unidirectional replication. Replication fork progression is significantly co-oriented with the transcription. Replication initiates stochastically, primarily within non-transcribed, broad (up to 150 kb) zones that often abut transcribed genes, and terminates dispersively between them. 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. ![]() Existing data have shown strong discrepancies. Despite intense investigation, human replication origins and termini remain elusive.
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