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Control of DNA replication in vitro using a reversible replication barrier

Nature Protocols (2024)Cite this article

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Abstract

A major obstacle to studying DNA replication is that it involves asynchronous and highly delocalized events. A reversible replication barrier overcomes this limitation and allows replication fork movement to be synchronized and localized, facilitating the study of replication fork function and replication coupled repair. Here we provide details on establishing a reversible replication barrier in vitro and using it to monitor different aspects of DNA replication. DNA template containing an array of lac operator (lacO) sequences is first bound to purified lac repressor (LacR). This substrate is then replicated in vitro using a biochemical replication system, which results in replication forks stalled on either side of the LacR array regardless of when or where they arise. Once replication forks are synchronized at the barrier, isopropyl-β-d-thiogalactopyranoside can be added to disrupt LacR binding so that replication forks synchronously resume synthesis. We describe how this approach can be employed to control replication fork elongation, termination, stalling and uncoupling, as well as assays that can be used to monitor these processes. We also explain how this approach can be adapted to control whether replication forks encounter a DNA lesion on the leading or lagging strand template and whether a converging fork is present. The required reagents can be prepared in 1–2 weeks and experiments using this approach are typically performed over 1–3 d. The main requirements for utilizing the LacR replication barrier are basic biochemical expertise and access to an in vitro system to study DNA replication. Investigators should also be trained in working with radioactive materials.

Key points

  • This protocol describes an approach to study DNA replication in vitro, based on the formation of a replication barrier that stalls asynchronously advancing replicative forks. Stalling of replication forks can be resolved by adding isopropyl-β-d-thiogalactopyranoside, causing the replication of the DNA template to restart synchronously.

  • Application of this method allows various aspects of DNA replication (elongation, termination, stalling and uncoupling) and replication-coupled DNA repair to be studied.

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Fig. 1: Control of replication fork movement by a lac repressor (LacR) array.
Fig. 2: Use of a LacR barrier to control replication fork termination, uncoupling and stalling.
Fig. 3: Purification of biotinylated LacR.
Fig. 4: Quality control of subcloned lacO array plasmids.
Fig. 5: Preparation of plasmids containing modified nucleotides.
Fig. 6: Replication fork uncoupling using a LacR barrier.
Fig. 7: Native gel analysis of replication fork structures.
Fig. 8: 2D gel analysis of replication fork structures.
Fig. 9: Denaturing gel analysis of nascent DNA strands.

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Data availability

Sequences for plasmid DNA templates (Table 1) are available upon request Source data are provided with this paper.

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Acknowledgements

We are grateful to J. Yeeles and K. Marians for the original LacR purification protocol and LacR expression plasmid. J.M.D. was supported by National Institutes of Health grant R01ES034847. E.J.V., S.N.D. and M.T.C. were supported by National Institutes of Health grant T32ES007028. M.T.C. was also supported by F32GM148024. Animal protocols were approved by Vanderbilt Division of Animal Care and Institutional Animal Care and Use committee. The authors complied with all relevant ethical regulations.

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  1. These authors contributed equally: Emma J. Vontalge, Tamar Kavlashvili.

Authors and Affiliations

  1. Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, USA

    Emma J. Vontalge, Tamar Kavlashvili, Steven N. Dahmen, Matthew T. Cranford & James M. Dewar

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  1. Emma J. Vontalge
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Contributions

J.M.D. developed the LacR barrier approach and related assays. J.M.D. and T.K. adopted the LacR barrier approach to induce site-specific uncoupling and developed related assays. J.M.D. and E.J.V. optimized and refined the LacR barrier approach to induce site-specific uncoupling and related assays. J.M.D., S.N.D. and M.T.C. developed the approach to introduce site-specific base modifications into template plasmid. E.J.V., T.K. and S.N.D. performed the experiments shown. J.M.D. and E.J.V. wrote the manuscript with input from S.N.D. and T.K.

Corresponding author

Correspondence to James M. Dewar.

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Key references using this protocol

Heintzman, D. R. et al. Cell Rep. 29, 422–436.e5 (2019): https://doi.org/10.1016/j.celrep.2019.08.097

Van Ravenstein, S. X. et al. EMBO J. 41, e110632 (2022): https://doi.org/10.15252/embj.2022110632

Kavlashvili, T. et al. Nat. Struct. Mol. Biol. 30, 115–124 (2023): https://doi.org/10.1038/s41594-022-00871-y

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Uncropped autoradiograph for Fig. 2b.

Source Data Fig. 3

Uncropped Coomassie-stained SDS–PAGE gel for Fig. 3b.

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Uncropped SYBR gold-stained agarose gel for Fig. 4b.

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Uncropped ethidium bromide-stained agarose gel for Fig. 5b.

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Uncropped autoradiograph for Fig. 6b.

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Uncropped autoradiograph for Fig. 7b.

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Uncropped autoradiographs for Fig. 8b and f.

Source Data Fig. 9

Uncropped autoradiograph for Fig. 9b.

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Vontalge, E.J., Kavlashvili, T., Dahmen, S.N. et al. Control of DNA replication in vitro using a reversible replication barrier. Nat Protoc (2024). https://doi.org/10.1038/s41596-024-00977-1

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