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A four-point molecular handover during Okazaki maturation

Nature Structural & Molecular Biology volume 30, pages 1505–1515 (2023)Cite this article

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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 polymerase, Pol I endonuclease and DNA 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 cryogenic-electron microscopy 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 a 5′ end that is a suitable substrate for the ligase activity of LigA once all RNA has been removed.

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Fig. 1: Comparison of Pol IIIαε and Pol I primer extension activity.
Fig. 2: Cryo-EM structures of Pol I bound to an upstream and downstream DNA substrate.
Fig. 3: Alternating activities of polymerase fingers and endonuclease domains.
Fig. 4: Pol I endonuclease cuts at the RNA–DNA junction.
Fig. 5: Ligase activity of the E. coli LigA and LigB.
Fig. 6: The β clamp does not appear to play a role in Okazaki fragment maturation.
Fig. 7: Schematic representation of Okazaki fragment maturation.

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Data availability

Cryo-EM maps and atomic models have been deposited in the Electron Microscopy Database and Protein Data Bank, respectively, under accession codes EMD-17033, PDB 8OOY, EMD-17005 and PDB 8OO6. Source data are provided with this paper. Other requests should be addressed to M.H.L. (m.h.lamers@lumc.nl).

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Acknowledgements

We thank the staff of the LUMC EM facility and the Netherlands Center for Electron Nanoscopy (NeCEN) for help with data collection and data processing. This work has been supported by an LUMC Research Fellowship to M.H.L. Access to NeCEN was supported by the Netherlands Electron Microscopy Infrastructure, project no. 184.034.014 of the National Roadmap for Large-Scale Research Infrastructure of the Dutch Research Council (NWO). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

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  1. Margherita M. Botto

    Present address: Department of Molecular and Cellular Biology, Geneva University, Geneva, Switzerland

  2. Alessandro Borsellini

    Present address: Department of Structural Biology, Human Technopole, Milan, Italy

Authors and Affiliations

  1. Department of Cell and Chemical Biology, Leiden University Medical Center (LUMC), Leiden, the Netherlands

    Margherita M. Botto, Alessandro Borsellini & Meindert H. Lamers

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Contributions

M.H.L. and M.M.B. conceived the overall experimental design. M.M.B. prepared samples, performed biochemical assays and analyzed data. A.B. collected and processed cryo-EM data. M.M.B. and M.H.L. wrote the manuscript with contributions from all authors.

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Correspondence to Meindert H. Lamers.

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Nature Structural & Molecular Biology thanks Nicholas Dixon, Mike O’Donnell and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Sara Osman and Dimitris Typas were the primary editors on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Cryo-EM data collection and data processing details.

a, Representative micrograph from the 11,000 micrographs collected. b, 2D class averages from full dataset. c, Fourier Shell Correlation between half-maps from final refinement. Green line: unmasked. Blue line: masked. Red line: phase randomized. Black line: corrected. Bottom (4th) panel shows the Fourier Shell Correlation of the model-to-map of the three structures (green line: full-length structure, blue line: closed structure, red line: open structure), d, Schematic representation of main data processing procedures. See Methods section for more details. e, Final map obtained after applying SuperEM code to Relion postprocessed map from dataset 1 and colored by local resolution. Color bar below the map shows the resolution range of the cryo-EM map in Å. f, Detail of model fitted to the final cryo-EM map from dataset 1. g, Orientational distribution of final set of refined particles from dataset 1. h, Anisotropy analysis of open and closed structure. Red line shows the half map FSC, with green lines representing the spread of the directional resolution defined by plus and minus one standard deviation from the mean. Blue bars show the histogram of one hundred directional resolutions evenly sampled of the 3D FSC. i, overlay of cryo-EM pol I structure with five X-ray crystallography structures of Pol I (pdb codes: 1l3t, 2bdp, 3tar, 6dsy, 7k50). Cryo-EM structure of open Pol I in cyan, with template DNA in black, extended primer in green, and displaced primer in red. All previously determined structures are shown in gray.

Extended Data Fig. 2 Positional modelling of the endonuclease domain in Pol I.

a, E. coli Pol I with predicted position of the endonuclease domain on top of the fingers domain, based on the position of the single-stranded flap (see also main Fig. 3a). b, Thermus aquaticus Pol I with its endonuclease domain adjacent to the 3′-5′ exonuclease domain33. c, Mycobacterium smegmatis Pol I with its endonuclease domain adjacent to the thumb domain31. d, Superposition of three Pol I endonuclease domains from T. aquaticus, M. smegmatis, and E. coli, (modelled using AlphaFold34). Red star marks the endonuclease active site. e, Superposition of E. coli Pol I endonuclease domain and T5 endonuclease bound to its substrate DNA35. The square marks the enlarged area shown below. Site of incision is marked by the two nucleotides in light blue. Two catalytic aspartates are shown in sticks. f, Similar comparison with human FEN136. Structures of T5 endonuclease and FEN1 were used to model the position of Pol I endonuclease in main Fig. 3b.

Extended Data Fig. 3 Pol I does not progress past a C in the template strand in absence of dGTP.

a, Pol I activity in absence of LigA using the templates shown in panel b, using only the three nucleotides dATP, dCTP, and dTTP. Experimental conditions are the same as in Fig. 5b. b, Substrates used for experiment in panel a.

Extended Data Fig. 4 The location of β-binding motifs of Pol I, LigA and Pol IIIα.

a, Model of Pol I and β-clamp. The β-clamp is shown in green surface, with binding pocket highlighted in yellow. The predicted β-binding motif of Pol I is shown in magenta sticks and is located in a helix of the thumb domain that interacts with the minor groove of the DNA. b, Model of LigA and β-clamp shown in two views rotated by 180°. The two predicted β-binding motifs are shown in magenta sticks. Motif LigA-1 is located in a helix that precedes a loop that interacts with the DNA. Motif LigA-2 is located on a strand that is part of the OB-domain that also interacts with the DNA. c, Cryo-EM structure of Pol IIIα bound to the β-clamp, exonuclease ε, and DNA52. The Pol IIIα β-binding motif is shown in magenta sticks and located on a loop at the end of the fingers domain and interacts with the binding pocket of the β-clamp. d, Alignment of β-binding motifs from Pol I, LigA and Pol IIIα that are highlighted in magenta in panels a-c.

Supplementary information

Supplementary Information

Supplementary Tables 1–3.

Reporting Summary

Peer Review File

Supplementary Video 1

Video showing a morph from closed to open cryo-EM map of Pol I.

Supplementary Video 2

Model of the single base pair translocation of the DNA in Pol I and the resulting displacement of a single nucleotide in the displaced strand.

Source data

Source Data Fig. 1

Unprocessed gels.

Source Data Fig. 3

Unprocessed gels.

Source Data Fig. 4

Unprocessed gels.

Source Data Fig. 5

Unprocessed gels.

Source Data Fig. 5

Statistical source data.

Source Data Fig. 6

Unprocessed gels.

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Botto, M.M., Borsellini, A. & Lamers, M.H. A four-point molecular handover during Okazaki maturation. Nat Struct Mol Biol 30, 1505–1515 (2023). https://doi.org/10.1038/s41594-023-01071-y

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