Genome-wide identification and characterisation of human DNA replication origins by initiation site sequencing (ini-seq)

Abstract

Next-generation sequencing has enabled the genome-wide identification of human DNA replication origins. However, different approaches to mapping replication origins, namely (i) sequencing isolated small nascent DNA strands (SNS-seq); (ii) sequencing replication bubbles (bubble-seq) and (iii) sequencing Okazaki fragments (OK-seq), show only limited concordance. To address this controversy, we describe here an independent high-resolution origin mapping technique that we call initiation site sequencing (ini-seq). In this approach, newly replicated DNA is directly labelled with digoxigenin-dUTP near the sites of its initiation in a cell-free system. The labelled DNA is then immunoprecipitated and genomic locations are determined by DNA sequencing. Using this technique we identify >25,000 discrete origin sites at sub-kilobase resolution on the human genome, with high concordance between biological replicates. Most activated origins identified by ini-seq are found at transcriptional start sites and contain G-quadruplex (G4) motifs. They tend to cluster in early-replicating domains, providing a correlation between early replication timing and local density of activated origins. Origins identified by ini-seq show highest concordance with sites identified by SNS-seq, followed by OK-seq and bubble-seq. Furthermore, germline origins identified by positive nucleotide distribution skew jumps overlap with origins identified by ini-seq and OK-seq more frequently and more specifically than do sites identified by either SNS-seq or bubble-seq.

Chromosomal Mcm2-7 distribution is the primary driver of the genome replication program in species from yeast to humans

“…On the one hand, the mammalian ORC reportedly does not exhibit sequence preferences but instead recognizes certain chromatin or DNA topological features (Remus et al, 2004;Vashee et al, 2003). On the other, several studies observed a bias toward GC-rich sequences in terms of both ORC localization and replication initiation (Cayrou et al, 2011;Dellino et al, 2013;Langley, Graf, Smith, & Krude, 2016;Miotto, Ji, & Struhl, 2016). Our observation that active and inactive X chromosomes, which differ vastly in chromatin organization but are almost identical in sequence, load MCM essentially identically favors the idea that DNA sequence or sequence-driven topological characteristics guide MCM distribution in mammals.…”

Replication dynamics of individual loci in single living cells reveal variation of stochasticity

“…Conversely, asynchronous replication of these regions, especially for late regions, may result from the asynchronous activation of different origins on the two chromosomes. The genome-wide detection of origins is mostly based on the identification of short nascent strands (SNSs) extracted from populations of millions of cells (Besnard et al, 2012;Cadoret et al, 2008;Cayrou et al, 2011;Langley et al, 2016;Picard et al, 2014;Sequeira-Mendes et al, 2009). Origin mapping in various species has shown that there are more origins in early-than in latereplicating regions, and that the origins in early-replicating regions are more efficient (Prioleau & MacAlpine, 2016).…”

Common fragile site instability in normal cells: Lessons and perspectives

“…47 In addition, two regions lacking initiation events have been identified within FRA6E. 47 In summary, late and incomplete replication of large genes is not per se a sufficient condition for chromosome fragility at CFSs, whereas the tissue specificity of several CFSs 9 may be associated with the plasticity characterizing, in mammalian cells, both DNA replication 48,[52][53][54] and establishment of replication timing. [55][56][57] Furthermore, nucleosome position as well as DNA-binding proteins acting as regulators of the epigenetic landscape at CFSs 58 and histone-specific modifications may account for the replication patterns observed at CFSs.…”