Determination of human DNA replication origin position and efficiency reveals principles of initiation zone organisation.
Journal
Nucleic acids research
ISSN: 1362-4962
Titre abrégé: Nucleic Acids Res
Pays: England
ID NLM: 0411011
Informations de publication
Date de publication:
22 07 2022
22 07 2022
Historique:
accepted:
20
06
2022
revised:
14
06
2022
received:
30
11
2021
pubmed:
9
7
2022
medline:
26
7
2022
entrez:
8
7
2022
Statut:
ppublish
Résumé
Replication of the human genome initiates within broad zones of ∼150 kb. The extent to which firing of individual DNA replication origins within initiation zones is spatially stochastic or localised at defined sites remains a matter of debate. A thorough characterisation of the dynamic activation of origins within initiation zones is hampered by the lack of a high-resolution map of both their position and efficiency. To address this shortcoming, we describe a modification of initiation site sequencing (ini-seq), based on density substitution. Newly replicated DNA is rendered 'heavy-light' (HL) by incorporation of BrdUTP while unreplicated DNA remains 'light-light' (LL). Replicated HL-DNA is separated from unreplicated LL-DNA by equilibrium density gradient centrifugation, then both fractions are subjected to massive parallel sequencing. This allows precise mapping of 23,905 replication origins simultaneously with an assignment of a replication initiation efficiency score to each. We show that origin firing within early initiation zones is not randomly distributed. Rather, origins are arranged hierarchically with a set of very highly efficient origins marking zone boundaries. We propose that these origins explain much of the early firing activity arising within initiation zones, helping to unify the concept of replication initiation zones with the identification of discrete replication origin sites.
Identifiants
pubmed: 35801867
pii: 6633902
doi: 10.1093/nar/gkac555
pmc: PMC9303276
doi:
Substances chimiques
DNA
9007-49-2
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
7436-7450Subventions
Organisme : Medical Research Council
ID : MC_U105178808
Pays : United Kingdom
Organisme : Medical Research Council
ID : U105178808
Pays : United Kingdom
Informations de copyright
© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.
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