DNA Molecular Combing-based Replication Fork Directionality Profiling

Overview

Journal Nucleic Acids Res

Publisher Oxford University Press

Specialty Biochemistry

Date 2021 Apr 9

PMID 33836085

Citations 7

Authors

Marion Blin

Laurent Lacroix

Nataliya Petryk

Yan Jaszczyszyn

Chun-Long Chen

Olivier Hyrien

Benoit Le Tallec

Affiliations

Soon will be listed here.

Abstract

The replication strategy of metazoan genomes is still unclear, mainly because definitive maps of replication origins are missing. High-throughput methods are based on population average and thus may exclusively identify efficient initiation sites, whereas inefficient origins go undetected. Single-molecule analyses of specific loci can detect both common and rare initiation events along the targeted regions. However, these usually concentrate on positioning individual events, which only gives an overview of the replication dynamics. Here, we computed the replication fork directionality (RFD) profiles of two large genes in different transcriptional states in chicken DT40 cells, namely untranscribed and transcribed DMD and CCSER1 expressed at WT levels or overexpressed, by aggregating hundreds of oriented replication tracks detected on individual DNA fibres stretched by molecular combing. These profiles reconstituted RFD domains composed of zones of initiation flanking a zone of termination originally observed in mammalian genomes and were highly consistent with independent population-averaging profiles generated by Okazaki fragment sequencing. Importantly, we demonstrate that inefficient origins do not appear as detectable RFD shifts, explaining why dispersed initiation has remained invisible to population-based assays. Our method can both generate quantitative profiles and identify discrete events, thereby constituting a comprehensive approach to study metazoan genome replication.

Citing Articles

The double life of mammalian DNA replication origins.

Hyrien O, Guilbaud G, Krude T Genes Dev. 2025; 39(5-6):304-324.

PMID: 39904559 PMC: 11874978. DOI: 10.1101/gad.352227.124.

Dual DNA replication modes: varying fork speeds and initiation rates within the spatial replication program in Xenopus.

Ciardo D, Haccard O, de Carli F, Hyrien O, Goldar A, Marheineke K Nucleic Acids Res. 2025; 53(3).

PMID: 39883014 PMC: 11781033. DOI: 10.1093/nar/gkaf007.

Monitoring and quantifying replication fork dynamics with high-throughput methods.

Fajri N, Petryk N Commun Biol. 2024; 7(1):729.

PMID: 38877080 PMC: 11178896. DOI: 10.1038/s42003-024-06412-1.

Starting DNA Synthesis: Initiation Processes during the Replication of Chromosomal DNA in Humans.

Nasheuer H, Meaney A Genes (Basel). 2024; 15(3).

PMID: 38540419 PMC: 10969946. DOI: 10.3390/genes15030360.

Neural network and kinetic modelling of human genome replication reveal replication origin locations and strengths.

Arbona J, Kabalane H, Barbier J, Goldar A, Hyrien O, Audit B PLoS Comput Biol. 2023; 19(5):e1011138.

PMID: 37253070 PMC: 10256156. DOI: 10.1371/journal.pcbi.1011138.

References

Hyrien O, Rappailles A, Guilbaud G, Baker A, Chen C, Goldar A . From simple bacterial and archaeal replicons to replication N/U-domains. J Mol Biol. 2013; 425(23):4673-89. DOI: 10.1016/j.jmb.2013.09.021. View

McGuffee S, Smith D, Whitehouse I . Quantitative, genome-wide analysis of eukaryotic replication initiation and termination. Mol Cell. 2013; 50(1):123-35. PMC: 3628276. DOI: 10.1016/j.molcel.2013.03.004. View

Urban J, Foulk M, Casella C, Gerbi S . The hunt for origins of DNA replication in multicellular eukaryotes. F1000Prime Rep. 2015; 7:30. PMC: 4371235. DOI: 10.12703/P7-30. View

Georgieva D, Liu Q, Wang K, Egli D . Detection of base analogs incorporated during DNA replication by nanopore sequencing. Nucleic Acids Res. 2020; 48(15):e88. PMC: 7470954. DOI: 10.1093/nar/gkaa517. View

de Carli F, Gaggioli V, Millot G, Hyrien O . Single-molecule, antibody-free fluorescent visualisation of replication tracts along barcoded DNA molecules. Int J Dev Biol. 2016; 60(7-8-9):297-304. DOI: 10.1387/ijdb.160139oh. View

Hennion M, Arbona J, Lacroix L, Cruaud C, Theulot B, Le Tallec B . FORK-seq: replication landscape of the Saccharomyces cerevisiae genome by nanopore sequencing. Genome Biol. 2020; 21(1):125. PMC: 7251829. DOI: 10.1186/s13059-020-02013-3. View

Audit B, Baker A, Chen C, Rappailles A, Guilbaud G, Julienne H . Multiscale analysis of genome-wide replication timing profiles using a wavelet-based signal-processing algorithm. Nat Protoc. 2012; 8(1):98-110. DOI: 10.1038/nprot.2012.145. View

Tubbs A, Sridharan S, van Wietmarschen N, Maman Y, Callen E, Stanlie A . Dual Roles of Poly(dA:dT) Tracts in Replication Initiation and Fork Collapse. Cell. 2018; 174(5):1127-1142.e19. PMC: 6591735. DOI: 10.1016/j.cell.2018.07.011. View

Mesner L, Valsakumar V, Cieslik M, Pickin R, Hamlin J, Bekiranov S . Bubble-seq analysis of the human genome reveals distinct chromatin-mediated mechanisms for regulating early- and late-firing origins. Genome Res. 2013; 23(11):1774-88. PMC: 3814878. DOI: 10.1101/gr.155218.113. View

10.

Lebofsky R, Heilig R, Sonnleitner M, Weissenbach J, Bensimon A . DNA replication origin interference increases the spacing between initiation events in human cells. Mol Biol Cell. 2006; 17(12):5337-45. PMC: 1679695. DOI: 10.1091/mbc.e06-04-0298. View

11.

Blin M, Le Tallec B, Nahse V, Schmidt M, Brossas C, Millot G . Transcription-dependent regulation of replication dynamics modulates genome stability. Nat Struct Mol Biol. 2019; 26(1):58-66. DOI: 10.1038/s41594-018-0170-1. View

12.

Besnard E, Babled A, Lapasset L, Milhavet O, Parrinello H, Dantec C . Unraveling cell type-specific and reprogrammable human replication origin signatures associated with G-quadruplex consensus motifs. Nat Struct Mol Biol. 2012; 19(8):837-44. DOI: 10.1038/nsmb.2339. View

13.

Guilbaud G, Rappailles A, Baker A, Chen C, Arneodo A, Goldar A . Evidence for sequential and increasing activation of replication origins along replication timing gradients in the human genome. PLoS Comput Biol. 2012; 7(12):e1002322. PMC: 3248390. DOI: 10.1371/journal.pcbi.1002322. View

14.

Daigaku Y, Keszthelyi A, Muller C, Miyabe I, Brooks T, Retkute R . A global profile of replicative polymerase usage. Nat Struct Mol Biol. 2015; 22(3):192-198. PMC: 4789492. DOI: 10.1038/nsmb.2962. View

15.

Muller C, Boemo M, Spingardi P, Kessler B, Kriaucionis S, Simpson J . Capturing the dynamics of genome replication on individual ultra-long nanopore sequence reads. Nat Methods. 2019; 16(5):429-436. PMC: 7617212. DOI: 10.1038/s41592-019-0394-y. View

16.

Kirstein N, Buschle A, Wu X, Krebs S, Blum H, Kremmer E . Human ORC/MCM density is low in active genes and correlates with replication time but does not delimit initiation zones. Elife. 2021; 10. PMC: 7993996. DOI: 10.7554/eLife.62161. View

17.

Demczuk A, Gauthier M, Veras I, Kosiyatrakul S, Schildkraut C, Busslinger M . Regulation of DNA replication within the immunoglobulin heavy-chain locus during B cell commitment. PLoS Biol. 2012; 10(7):e1001360. PMC: 3393677. DOI: 10.1371/journal.pbio.1001360. View

18.

Foss E, Sripathy S, Gatbonton-Schwager T, Kwak H, Thiesen A, Lao U . Chromosomal Mcm2-7 distribution and the genome replication program in species from yeast to humans. PLoS Genet. 2021; 17(9):e1009714. PMC: 8443269. DOI: 10.1371/journal.pgen.1009714. View

19.

Cadoret J, Meisch F, Hassan-Zadeh V, Luyten I, Guillet C, Duret L . Genome-wide studies highlight indirect links between human replication origins and gene regulation. Proc Natl Acad Sci U S A. 2008; 105(41):15837-42. PMC: 2572913. DOI: 10.1073/pnas.0805208105. View

20.

Cayrou C, Coulombe P, Vigneron A, Stanojcic S, Ganier O, Peiffer I . Genome-scale analysis of metazoan replication origins reveals their organization in specific but flexible sites defined by conserved features. Genome Res. 2011; 21(9):1438-49. PMC: 3166829. DOI: 10.1101/gr.121830.111. View