Integrative analysis of DNA replication origins and ORC/MCM binding sites in human cells reveals a lack of overlap

Abstract

DNA replication initiates from ~50,000 origins on human chromosomes in each cell-cycle and the origins are hypothesized to be specified by binding of factors like the Origin Recognition Complex (ORC) or CTCF or other features like G-quadruplexes. We have performed an integrative analysis of 113 genome-wide human origin profiles (from five different techniques) and 5 ORC-binding site datasets to critically evaluate whether the most reproducible origins are specified by these features. Out of ~7.5 million 300 bp chromosomal fragments reported to harbor origins by all the datasets, only 0.27% were reproducibly detected by four techniques (20,250 shared origins), suggesting extensive variability in origin usage and identification in different circumstances. 21% of the shared origins overlap with transcriptional promoters, posing a conundrum. Although the shared origins overlap more than union origins with constitutive CTCF binding sites, G-quadruplex sites and activating histone marks, these overlaps are comparable or less than that of known Transcription Start Sites, so that these features could be enriched in origins because of the overlap of origins with epigenetically open, promoter-like sequences. Only 6.4% of the 20,250 shared origins were within 1 kb from any of the ~13,000 reproducible ORC binding sites in human cancer cells, in contrast to the nearly 100% overlap between the two in the yeast, S. cerevisiae. Thus, in human cancer cell-lines, replication origins appear to be specified by highly variable stochastic events dependent on the high epigenetic accessibility around promoters, without extensive overlap between the most reproducible origins and ORC-binding sites.

Chromatin’s Influence on Pre-Replication Complex Assembly and Function

“…The Dutta and Zang labs have recently suggested that the origins are specified by diverse stochastic events that are dependent on the epigenetic accessibility around promoters. However, intriguingly, the ORC-binding sites or the MCM complex did not overlap with the origins [ 49 ]. A series of genome-wide studies with various ORC antibodies, specifically at distinct time points within G1, would be important to pinpoint ORC dynamics in human cells.…”

The Origin Recognition Complex: From Origin Selection to Replication Licensing in Yeast and Humans

“…In these inverted ORC-binding sites in proximity, a MCM double hexamer is recruited at or very near to the site where ORC is bound, and replication initiation takes place. This mechanism is unlikely to work in the case of humans where replication origins are devoid of conserved sequence motifs in proximity specific for ORC binding [59,60]. Without a second binding site in proximity, wherever ORC binds, it will only recruit a single MCM hexamer [61].…”

R-loops acted on by RNase H1 are a determinant of chromosome length-associated DNA replication timing and genome stability inLeishmania

“…In the larger genomes of mammals, identification of origins has proved more challenging due to a lack of consistency between datasets that map DNA replication initiation, or localisation of ORC 1, 16 . In part, such inconsistency may be due to mammalian origins not being sequence-conserved genome features, but could also reflect greater use of stochastic, potentially ORC-independent initiation than is seen in yeast 17–21 . Nonetheless, mammalian replication timing appears to be organised over several scales.…”