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PreprintCited by 9DNA Repair MechanismsEnzyme Structure and FunctionBacterial Genetics and Biotechnology

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A four-point molecular handover during Okazaki maturation

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Botto, Margherita M., Borsellini, Alessandro, Lamers, Meindert H.·2022·Nature Structural & Molecular Biology
DOI 10.1101/2022.08.02.502465

Preprint — published as 10.1038/s41594-023-01071-y · Paper page

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Abstract

DNA replication introduces thousands of RNA primers into the lagging strand that need to be removed for replication to be completed. In Escherichia coli when the replicative DNA polymerase Pol IIIα terminates at a previously synthesized RNA primer, DNA Pol I takes over and continues DNA synthesis while displacing the downstream RNA primer. The displaced primer is subsequently excised by an endonuclease, followed by the sealing of the nick by a DNA ligase. Yet how the sequential actions of Pol IIIα, Pol I, endonuclease and ligase are coordinated is poorly defined. Here we show that each enzymatic activity prepares the DNA substrate for the next activity, creating an efficient four-point molecular handover. The cryo-EM structure of Pol I bound to a DNA substrate with both an upstream and downstream primer reveals how it displaces the primer in a manner analogous to the monomeric helicases. Moreover, we find that in addition to its flap-directed nuclease activity, the endonuclease domain of Pol I also specifically cuts at the RNA/DNA junction, thus marking the end of the RNA primer and creating an 5’ end that is a suitable substrate for the ligase activity of LigA once all RNA has been removed.

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Metadata

Authors
Botto, M. M., Borsellini, A., & Lamers, M. H.
Affiliations
  • University of Geneva (CH)
  • Leiden University Medical Center (NL)
Corresponding author
Lamers, Meindert H.
ORCID
Venue
Nature Structural & Molecular Biology
Publisher
openRxiv
Published
2022-08-02
Volume
30
Issue
10
Pages
1505-1515
ISSN
1545-9985 (ISSN-L)
DOI
10.1101/2022.08.02.502465
OpenAlex
W4386142497
Cited by
9
References
1
Citations by year
2026: 5 · 2025: 3 · 2024: 1
FWCI
0.80
Type
journal article
Language
en
Open access
Licence
cc-by-nc-nd
Locations
  • Nature Structural & Molecular Biology (publishedVersion)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • Leiden Repository (Leiden University) (submittedVersion)
Funders
  • Nederlandse Organisatie voor Wetenschappelijk Onderzoek (184.034.014)
  • Leids Universitair Medisch Centrum
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA Repair Mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Structural Biology › Enzyme Structure and Function
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
Keywords
Okazaki fragment maturation, RNA primer removal, DNA polymerase I, lagging strand synthesis, Escherichia coli DNA replication, flap endonuclease, cryo-EM structure
MeSH terms
DNA Ligase ATP, DNA, DNA Polymerase III, DNA Replication, Endonucleases, DNA Ligases, RNA

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