Control of DNA replication in vitro using a reversible replication barrier

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

Published: 2024-04-09 DOI: 10.1038/s41596-024-00977-1

DNA replication

Reversible replication barrier

In vitro

Replication fork

Lac repressor

AI 解读

Deoxyfluorination of phenols for chemoselective18F-labeling of peptides

Stable isotope tracing to assess tumor metabolism in vivo

Measuring DNA modifications with the comet assay: a compendium of protocols

Detect-seq, a chemical labeling and biotin pull-down approach for the unbiased and genome-wide off-target evaluation of programmable cytosine base editors

A systems-level mass spectrometry-based technique for accurate and sensitive quantification of the RNA cap epitranscriptome

Spooling electrochemiluminescence spectroscopy: development, applications and beyond

Chronic, cortex-wide imaging of specific cell populations during behavior

Chromatin accessibility profiling by ATAC-seq

Global analysis of RNA-binding protein dynamics by comparative and enhanced RNA interactome capture

Pooled genetic perturbation screens with image-based phenotypes

查看全部

Sections

Figures

References

Introduction
Materials
Procedure 1: preparation of materials
Procedure 2: experimental approaches
- In vitro DNA replication using LacR as a reversible replication barrier
- Separation of replication intermediates using native agarose gels
Procedure 3: analytical gels
Procedure 4: data acquisition and analysis
- Phosphorimaging
- ImageJ analysis
Troubleshooting
Timing
Anticipated results
Acknowledgements

Jackson, D. A. S-phase progression in synchronized human cells. Exp. Cell Res. 220, 62–70 (1995).
Berti, M., Cortez, D. & Lopes, M. The plasticity of DNA replication forks in response to clinically relevant genotoxic stress. Nat. Rev. Mol. Cell Biol. 21, 633–651 (2020).
Chen, Y. H. et al. Transcription shapes DNA replication initiation and termination in human cells. Nat. Struct. Mol. Biol. 26, 67–77 (2019).
Czajkowsky, D. M., Liu, J., Hamlin, J. L. & Shao, Z. DNA combing reveals intrinsic temporal disorder in the replication of yeast chromosome VI. J. Mol. Biol. 375, 12–19 (2008).
McGuffee, S. R., Smith, D. J. & Whitehouse, I. Quantitative, genome-wide analysis of eukaryotic replication initiation and termination. Mol. Cell 50, 123–135 (2013).
Wang, W. et al. Genome-wide mapping of human DNA replication by optical replication mapping supports a stochastic model of eukaryotic replication. Mol. Cell 81, 2975–2988.e2976 (2021).
Baris, Y., Taylor, M. R. G., Aria, V. & Yeeles, J. T. P. Fast and efficient DNA replication with purified human proteins. Nature 606, 204–210 (2022).
Walter, J., Sun, L. & Newport, J. Regulated chromosomal DNA replication in the absence of a nucleus. Mol. Cell 1, 519–529 (1998).
Yeeles, J. T., Deegan, T. D., Janska, A., Early, A. & Diffley, J. F. Regulated eukaryotic DNA replication origin firing with purified proteins. Nature 519, 431–435 (2015).
Loveland, A. B., Habuchi, S., Walter, J. C. & van Oijen, A. M. A general approach to break the concentration barrier in single-molecule imaging. Nat. Methods 9, 987–992 (2012).
Yardimci, H., Loveland, A. B., Habuchi, S., van Oijen, A. M. & Walter, J. C. Uncoupling of sister replisomes during eukaryotic DNA replication. Mol. Cell 40, 834–840 (2010).
Emerson, D. J. et al. Cohesin-mediated loop anchors confine the locations of human replication origins. Nature 606, 812–819 (2022).
Dewar, J. M., Budzowska, M. & Walter, J. C. The mechanism of DNA replication termination in vertebrates. Nature 525, 345–350 (2015).
Low, E., Chistol, G., Zaher, M. S., Kochenova, O. V. & Walter, J. C. The DNA replication fork suppresses CMG unloading from chromatin before termination. Genes Dev. 34, 1534–1545 (2020).
Payne, B. T. et al. Replication fork blockage by transcription factor–DNA complexes in Escherichia coli. Nucleic Acids Res. 34, 5194–5202 (2006).
Possoz, C., Filipe, S. R., Grainge, I. & Sherratt, D. J. Tracking of controlled Escherichia coli replication fork stalling and restart at repressor-bound DNA in vivo. EMBO J. 25, 2596–2604 (2006).
Vrtis, K. B. et al. Single-strand DNA breaks cause replisome disassembly. Mol. Cell 81, 1309–1318.e1306 (2021).
Zhang, J. et al. DNA interstrand cross-link repair requires replication-fork convergence. Nat. Struct. Mol. Biol. 22, 242–247 (2015).
Duxin, J. P., Dewar, J. M., Yardimci, H. & Walter, J. C. Repair of a DNA–protein crosslink by replication-coupled proteolysis. Cell 159, 346–357 (2014).
Semlow, D. R., Zhang, J., Budzowska, M., Drohat, A. C. & Walter, J. C. Replication-dependent unhooking of DNA interstrand cross-links by the NEIL3 glycosylase. Cell 167, 498–511.e414 (2016).
Campos, L. V. et al. RTEL1 and MCM10 overcome topological stress during vertebrate replication termination. Cell Rep. 42, 112109 (2023).
Sparks, J. L. et al. The CMG helicase bypasses DNA-protein cross-links to facilitate their repair. Cell 176, 167–181.e121 (2019).
Van Ravenstein, S. X. et al. Topoisomerase II poisons inhibit vertebrate DNA replication through distinct mechanisms. EMBO J. 41, e110632 (2022).
Kavlashvili, T., Liu, W., Mohamed, T. M., Cortez, D. & Dewar, J. M. Replication fork uncoupling causes nascent strand degradation and fork reversal. Nat. Struct. Mol. Biol. 30, 115–124 (2023).
Deng, L. et al. Mitotic CDK promotes replisome disassembly, fork breakage, and complex DNA rearrangements. Mol. Cell 73, 915–929.e916 (2019).
Dewar, J. M., Low, E., Mann, M., Räschle, M. & Walter, J. C. CRL2(Lrr1) promotes unloading of the vertebrate replisome from chromatin during replication termination. Genes Dev. 31, 275–290 (2017).
Heintzman, D. R., Campos, L. V., Byl, J. A. W., Osheroff, N. & Dewar, J. M. Topoisomerase II is crucial for fork convergence during vertebrate replication termination. Cell Rep. 29, 422–436.e425 (2019).
Sato, K., Martin-Pintado, N., Post, H., Altelaar, M. & Knipscheer, P. Multistep mechanism of G-quadruplex resolution during DNA replication. Sci. Adv. 7, eabf8653 (2021).
Räschle, M. et al. Mechanism of replication-coupled DNA interstrand crosslink repair. Cell 134, 969–980 (2008).
Lebofsky, R., Takahashi, T. & Walter, J. C. DNA replication in nucleus-free Xenopus egg extracts. Methods Mol. Biol. 521, 229–252 (2009).
Klein Douwel, D. et al. XPF-ERCC1 acts in unhooking DNA interstrand crosslinks in cooperation with FANCD2 and FANCP/SLX4. Mol. Cell. 54, 460–471 (2014).
Knipscheer, P. et al. The Fanconi anemia pathway promotes replication-dependent DNA interstrand cross-link repair. Science 326, 1698–1701 (2009).
Budzowska, M., Graham, T. G., Sobeck, A., Waga, S. & Walter, J. C. Regulation of the Rev1–pol ζ complex during bypass of a DNA interstrand cross-link. EMBO J. 34, 1971–1985 (2015).
Long, D. T., Joukov, V., Budzowska, M. & Walter, J. C. BRCA1 promotes unloading of the CMG helicase from a stalled DNA replication fork. Mol. Cell 56, 174–185 (2014).
Räschle, M. et al. DNA repair. Proteomics reveals dynamic assembly of repair complexes during bypass of DNA cross-links. Science 348, 1253671 (2015).
Larsen, N. B. et al. Replication-coupled DNA–protein crosslink repair by SPRTN and the proteasome in Xenopus egg extracts. Mol. Cell 73, 574–588.e577 (2019).
Yeeles, J. T. P., Janska, A., Early, A. & Diffley, J. F. X. How the Eukaryotic replisome achieves rapid and efficient DNA replication. Mol. Cell 65, 105–116 (2017).
Minamino, M., Bouchoux, C., Canal, B., Diffley, J. F. X. & Uhlmann, F. A replication fork determinant for the establishment of sister chromatid cohesion. Cell 186, 837–849.e811 (2023).
Casas-Delucchi, C. S., Daza-Martin, M., Williams, S. L. & Coster, G. The mechanism of replication stalling and recovery within repetitive DNA. Nat. Commun. 13, 3953 (2022).
Chacin, E. et al. Establishment and function of chromatin organization at replication origins. Nature 616, 836–842 (2023).
Douglas, M. E. & Diffley, J. F. X. Budding yeast Rap1, but not telomeric DNA, is inhibitory for multiple stages of DNA replication in vitro. Nucleic Acids Res. 49, 5671–5683 (2021).
Devbhandari, S. & Remus, D. Rad53 limits CMG helicase uncoupling from DNA synthesis at replication forks. Nat. Struct. Mol. Biol. 27, 461–471 (2020).
Baretić, D. et al. Cryo-EM structure of the fork protection complex bound to CMG at a replication fork. Mol. Cell 78, 926–940.e913 (2020).
Jenkyn-Bedford, M. et al. A conserved mechanism for regulating replisome disassembly in eukaryotes. Nature 600, 743–747 (2021).
Peter, B. J., Ullsperger, C., Hiasa, H., Marians, K. J. & Cozzarelli, N. R. The structure of supercoiled intermediates in DNA replication. Cell 94, 819–827 (1998).
Suski, C. & Marians, K. J. Resolution of converging replication forks by RecQ and topoisomerase III. Mol. Cell 30, 779–789 (2008).
Larsen, N. B., Sass, E., Suski, C., Mankouri, H. W. & Hickson, I. D. The Escherichia coli Tus–Ter replication fork barrier causes site-specific DNA replication perturbation in yeast. Nat. Commun. 5, 3574 (2014).
Willis, N. A. et al. BRCA1 controls homologous recombination at Tus/Ter-stalled mammalian replication forks. Nature 510, 556–559 (2014).
Elango, R. et al. The structure-specific endonuclease complex SLX4–XPF regulates Tus–Ter-induced homologous recombination. Nat. Struct. Mol. Biol. 29, 801–812 (2022).
Gallina, I. et al. The ubiquitin ligase RFWD3 is required for translesion DNA synthesis. Mol. Cell 81, 442–458.e449 (2021).
Kose, H. B., Larsen, N. B., Duxin, J. P. & Yardimci, H. Dynamics of the Eukaryotic replicative helicase at lagging-strand protein barriers support the steric exclusion model. Cell Rep. 26, 2113–2125.e2116 (2019).
Hizume, K., Endo, S., Muramatsu, S., Kobayashi, T. & Araki, H. DNA polymerase ε-dependent modulation of the pausing property of the CMG helicase at the barrier. Genes Dev. 32, 1315–1320 (2018).
Beuzer, P., Quivy, J. P. & Almouzni, G. Establishment of a replication fork barrier following induction of DNA binding in mammalian cells. Cell Cycle 13, 1607–1616 (2014).
Roukos, V., Burgess, R. C. & Misteli, T. Generation of cell-based systems to visualize chromosome damage and translocations in living cells. Nat. Protoc. 9, 2476–2492 (2014).
Hua, X. H. & Newport, J. Identification of a preinitiation step in DNA replication that is independent of origin recognition complex and cdc6, but dependent on cdk2. J. Cell Biol. 140, 271–281 (1998).
Hua, X. H., Yan, H. & Newport, J. A role for Cdk2 kinase in negatively regulating DNA replication during S phase of the cell cycle. J. Cell Biol. 137, 183–192 (1997).
Sparks, J. & Walter, J. C. Extracts for analysis of DNA replication in a nucleus-free system. Cold Spring Harb. Protoc. https://doi.org/10.1101/pdb.prot097154 (2019).
Sheehan, M. A., Mills, A. D., Sleeman, A. M., Laskey, R. A. & Blow, J. J. Steps in the assembly of replication-competent nuclei in a cell-free system from Xenopus eggs. J. Cell Biol. 106, 1–12 (1988).
Santamaría, D. et al. Bi-directional replication and random termination. Nucleic Acids Res. 28, 2099–2107 (2000).
Yardimci, H. et al. Bypass of a protein barrier by a replicative DNA helicase. Nature 492, 205–209 (2012).
Laskey, R. A., Mills, A. D. & Morris, N. R. Assembly of SV40 chromatin in a cell-free system from Xenopus eggs. Cell 10, 237–243 (1977).
Amunugama, R. et al. Replication fork reversal during DNA interstrand crosslink repair requires CMG unloading. Cell Rep. 23, 3419–3428 (2018).
Long, D. T., Räschle, M., Joukov, V. & Walter, J. C. Mechanism of RAD51-dependent DNA interstrand cross-link repair. Science 333, 84–87 (2011).

Control of DNA replication in vitro using a reversible replication barrier

推荐阅读