High-resolution Repli-Seq defines the temporal choreography of initiation, elongation and termination of replication in mammalian cells

Abstract

Background: DNA replication in mammalian cells occurs in a defined temporal order during S phase, known as the replication timing (RT) programme. Replication timing is developmentally regulated and correlated with chromatin conformation and local transcriptional potential. Here, we present RT profiles of unprecedented temporal resolution in two human embryonic stem cell lines, human colon carcinoma line HCT116, and mouse embryonic stem cells and their neural progenitor derivatives. Results: Fine temporal windows revealed a remarkable degree of cell-to-cell conservation in RT, particularly at the very beginning and ends of S phase, and identified 5 temporal patterns of replication in all cell types, consistent with varying degrees of initiation efficiency. Zones of replication initiation (IZs) were detected throughout S phase and interacted in 3D space preferentially with other IZs of similar firing time. Temporal transition regions were resolved into segments of uni-directional replication punctuated at specific sites by small, inefficient IZs. Sites of convergent replication were divided into sites of termination or large constant timing regions consisting of many synchronous IZs in tandem. Developmental transitions in RT occured mainly by activating or inactivating individual IZs or occasionally by altering IZ firing time, demonstrating that IZs, rather than individual origins, are the units of developmental regulation. Finally, haplotype phasing revealed numerous regions of allele-specific and alleleindependent asynchronous replication. Allele-independent asynchronous replication was correlated with the presence of previously mapped common fragile sites. Conclusions: Altogether, these data provide a detailed temporal choreography of DNA replication in mammalian cells.

High-throughput analysis of single human cells reveals the complex nature of DNA replication timing control

“…While it is conceivably straightforward to envision how measurements with limited resolution would give the impression of domains or CTRs where none exist, it appears more difficult to reconcile the sharp and discrete initiation peaks in our single cell data with the idea of large regions with constant replication timing. In contrast, our data is consistent with recent high-resolution studies that suggest that replication initiation is confined to regions of several tens of kilobases 5,10 , although we do not find compelling evidence for widespread ectopic initiation from region outside commonly used initiation sites 5 . While many previous studies of mammalian replication origins relied on biochemical enrichments of DNA synthesis events 1 , single-cell DNA sequencing more reliably represents productive and internally-validated DNA replication.…”

Genome-wide analysis of DNA replication and DNA double-strand breaks using TrAEL-seq

“…Furthermore, comparison to GLOE-seq results from a LIG1-depleted human cell line that is defective in Okazaki fragment ligation again revealed a striking similarity to the hESC TrAEL-seq data, although with the opposite polarity ( Fig 3H and S3H Fig ) [ 40 ]. Interestingly, a subset of origins were reproducibly detected in hESC samples but absent in the HCT116 data, consistent with evidence that origin usage differs between these cell lines ( Fig 3H , green arrows) [ 24 ].…”

“…Many methods for mapping DNA replication have been developed, which can be broadly divided into those which measure copy number changes through S-phase and those which analyse replication forks or replication bubbles directly. Copy number analysis stratifies the genome based on replication timing and defines early and late-firing origins [ 24 – 27 ]. This requires segregation of cell populations at different stages of replication or between replicating and non-replicating cells, either by cell cycle synchronisation or, more flexibly, by flow cytometry.…”

Human ORC/MCM density is low in active genes and correlates with replication time but does not solely define replication initiation zones

“…Within the EdUseq-HU zones, the most efficient sites were associated with poly(dA:dT) tracts but not any of the GC-rich motifs found by SNS-seq (Tubbs et al, 2018). Repli-seq IZs, which are highly consistent with OK-seq IZs, were also recently identified in high-resolution replication timing (RT) profiles (Zhao et al, 2020).…”