Genome-wide measurement of DNA replication fork directionality and quantification of DNA replication initiation and termination with Okazaki fragment sequencing

Wu, Xia; Liu, Yaqun; d’Aubenton-Carafa, Yves; Thermes, Claude; Hyrien, Olivier; Chen, Chun-Long; Petryk, Nataliya

doi:10.1038/s41596-022-00793-5

Cited by 4 publications

(2 citation statements)

References 98 publications

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“…We used the OK-seq technique 38 to map initiation zones (IZs), referred hereafter as constitutive OK-origins, in untreated JEFF cells and compared the localization of those origins to that of ATRAP peaks. We identified 7963 constitutive OK-origins across the whole genome, 5656 lying in early replicating domains (Fig.…”

Section: Atrap Peaks Co-map With Early Ok-seq Initiation Zonesmentioning

confidence: 99%

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The balance between ATR and DDK activities controls TopBP1-mediated locking of dormant origins at the pre-IC stage

Koundrioukoff,

Kim,

Alary

et al. 2023

Preprint

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SummaryReplication stress, a major hallmark of cancers, and ensuing genome instability source from impaired progression of replication forks. The first line of defense against fork slowing is compensation, a long-described process that elicits firing of otherwise dormant origins. It remains unclear whether compensation requires activation of the DNA replication checkpoint or passively results from lengthening of the window of time during which dormant origins can fire when fork progression slows, or both. Using molecular DNA combing we show here that a linear relationship ties inter-origin distances to fork speeds, independently of the checkpoint status. We called this line “stressline” and further show that its slope enables precise quantification of the compensation efficiency. Comparison of the slopes in different genetic backgrounds reveals that compensation requires ATR, not CHK1, while TopBP1 and CDC7/DBF4 repress dormant origin activation. These results strongly suggest that TopBP1 locks dormant origins at the pre-IC stage and that ATR and DDK oppose to control the conversion of dormant pre-ICs into functional salvage origins. Both passive and active processes thus contribute to compensation. Moreover, Repli-seq and OK-seq analyses confirm the activating role of ATR and permit development of ATRAP-seq, a new procedure allowing mapping of early constitutive origins.

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Section: Atrap Peaks Co-map With Early Ok-seq Initiation Zonesmentioning

confidence: 99%

“…OK-seq libraries were generated as previously described 38 . Libraries were sequenced on an Illumina NextSeq 500 sequencing system using Paired-end (75 cycles).…”

Section: Ok-seq Data Generation and Processingmentioning

confidence: 99%

The balance between ATR and DDK activities controls TopBP1-mediated locking of dormant origins at the pre-IC stage

Koundrioukoff,

Kim,

Alary

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

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Regulation of replication timing inSaccharomyces cerevisiae

Berners-Lee,

Gilmore,

Berkemeier

et al. 2024

Preprint

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In order to maintain genomic integrity, DNA replication must be highly coordinated. Disruptions in this process can cause replication stress which is aberrant in many pathologies including cancer. Despite this, little is known about the mechanisms governing the temporal regulation of DNA replication initiation, thought to be related to the limited copy number of firing factors. Here, we present a high (1-kilobase) resolution stochastic model of Saccharomyces cerevisiae whole-genome replication in which origins compete to associate with limited firing factors. After developing an algorithm to fit this model to replication timing data, we validated the model by reproducing experimental inter-origin distances, origin efficiencies, and replication fork directionality. This suggests the model accurately simulates the aspects of DNA replication most important for determining its dynamics. We also use the model to predict measures of DNA replication dynamics which are yet to be determined experimentally and investigate the potential impacts of variations in firing factor concentrations on DNA replication.

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Nuclear DNA replication inLeishmania majorrelies on a single constitutive origin per chromosome supplemented by thousands of stochastic initiation events

Damasceno,

Silva,

Marques

et al. 2024

Preprint

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Understanding genome duplication requires characterisation of the locations where DNA replication initiates, termed origins. Genome-wide mapping of DNA replication origins has mainly been derived from population-based techniques, with only a few studies examining origin location and usage at the single-cell or single-molecule level.Leishmaniaare protozoan parasites where the first attempt to map DNA replication suggested the unprecedented use, for a eukaryote, of just a single origin per chromosome, while a subsequent approach suggested around 200-fold more origins. To reconcile these data and understand DNA replication dynamics inLeishmania major, we have applied DNAscent, a deep learning assay that uses long-read Nanopore sequencing to detect patterns of BrdU incorporation in individual DNA molecules, allowing the description of DNA replication fork movement and prediction of initiation and termination sites across the parasite genome. Our findings confirm the pre-eminence of a single locus of DNA replication initiation in each chromosome and reveal that this locus alone is constitutively activated in S-phase, with bidirectional forks emerging from discrete sites at the ends of multigene transcription units. DNAscent also reveals a much larger number of DNA replication initiation events that have not been detected in any previous mapping and are used stochastically, but whose abundance is greater as chromosome size increases. We show that each of these stochastic initiation sites localise to regions with high AT content, increased G-quadruplex levels and lower chromatin occupancy. In addition, we find markedly increased stochastic DNA replication initiation at sites with lower levels of nascent RNA transcripts. Finally, we show that all DNA replication initiation events result in mutagenesis. This work reveals a novel, bimodal strategy for DNA replication programming inLeishmaniathat drives genome transmission, replication timing and variation.

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Genome-wide measurement of DNA replication fork directionality and quantification of DNA replication initiation and termination with Okazaki fragment sequencing

Cited by 4 publications

References 98 publications

The balance between ATR and DDK activities controls TopBP1-mediated locking of dormant origins at the pre-IC stage

The balance between ATR and DDK activities controls TopBP1-mediated locking of dormant origins at the pre-IC stage

Regulation of replication timing inSaccharomyces cerevisiae

Nuclear DNA replication inLeishmania majorrelies on a single constitutive origin per chromosome supplemented by thousands of stochastic initiation events

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