Carrington Jamie T, Wilson Rosemary H C, de La Vega Eduardo, Thiyagarajan Sathish, Barker Tom, Catchpole Leah, Durrant Alex, Knitlhoffer Vanda, Watkins Chris, Gharbi Karim, Nieduszynski Conrad A
University of Oxford, Oxford, UK.
Cardiff University, Cardiff, UK.
Genome Biol. 2025 May 9;26(1):122. doi: 10.1186/s13059-025-03591-w.
The identification of sites of DNA replication initiation in mammalian cells has been challenging. Here, we present unbiased detection of replication initiation events in human cells using BrdU incorporation and single-molecule nanopore sequencing.
Increases in BrdU incorporation allow us to measure DNA replication dynamics, including identification of replication initiation, fork direction, and termination on individual nanopore sequencing reads. Importantly, initiation and termination events are identified on single molecules with high resolution, throughout S-phase, genome-wide, and at high coverage at specific loci using targeted enrichment. We find a significant enrichment of initiation sites within the broad initiation zones identified by population-level studies. However, these focused initiation sites only account for ~ 20% of all identified replication initiation events. Most initiation events are dispersed throughout the genome and are missed by cell population approaches. This indicates that most initiation occurs at sites that, individually, are rarely used. These dispersed initiation sites contrast with the focused sites identified by population studies, in that they do not show a strong relationship to transcription or a particular epigenetic signature.
We show here that single-molecule sequencing enables unbiased detection and characterization of DNA replication initiation events, including the numerous dispersed initiation events that replicate most of the human genome.
在哺乳动物细胞中鉴定DNA复制起始位点一直具有挑战性。在此，我们使用BrdU掺入和单分子纳米孔测序技术对人类细胞中的复制起始事件进行无偏检测。
BrdU掺入量的增加使我们能够测量DNA复制动态，包括在单个纳米孔测序读数上鉴定复制起始、叉方向和终止。重要的是，在整个S期、全基因组范围内以及使用靶向富集在特定基因座上以高覆盖率在单分子上鉴定起始和终止事件。我们发现在群体水平研究确定的广泛起始区域内起始位点显著富集。然而，这些集中的起始位点仅占所有已鉴定复制起始事件的约20%。大多数起始事件分散在整个基因组中，细胞群体方法会遗漏这些事件。这表明大多数起始发生在单个很少使用的位点。这些分散的起始位点与群体研究确定的集中位点形成对比，因为它们与转录或特定表观遗传特征没有很强的关系。
我们在此表明，单分子测序能够对DNA复制起始事件进行无偏检测和表征，包括复制大部分人类基因组的众多分散起始事件。
