Understanding the mechanisms of replication stress response following genotoxic stress induction is rapidly emerging as a central theme in cell survival and human disease. The DNA fiber assay is one of the most powerful tools to study alterations in replication fork dynamics genome-wide at single-molecule resolution. This approach relies on the ability of many organisms to incorporate thymidine analogs into replicating DNA and is widely used to study how genotoxic agents perturb DNA replication. Here, we review different approaches available to prepare DNA fibers and discuss important limitations of each approach. We also review how DNA fiber analysis can be used to shed light upon several replication parameters including fork progression, restart, termination, and new origin firing. Next, we discuss a modified DNA fiber protocol to monitor the presence of single-stranded DNA (ssDNA) gaps on ongoing replication forks. ssDNA gaps are very common intermediates of several replication stress response mechanisms, but they cannot be detected by standard DNA fiber approaches due to the resolution limits of this technique. We discuss a novel strategy that relies on the use of an ssDNA-specific endonuclease to nick the ssDNA gaps and generate shorter DNA fibers that can be used as readout for the presence of ssDNA gaps. Finally, we describe a follow-up DNA fiber approach that can be used to study how ssDNA gaps are repaired postreplicatively.
Keywords: DNA fiber analysis; DNA replication; Postreplication repair; Replication stress; S1 nuclease; Single-stranded DNA gap.
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Martins DJ, Tirman S, Quinet A, Menck CFM. Martins DJ, et al. J Vis Exp. 2022 Feb 3;(180). doi: 10.3791/63448. J Vis Exp. 2022. PMID: 35188138
Vindigni A, Lopes M. Vindigni A, et al. Biophys Chem. 2017 Jun;225:3-9. doi: 10.1016/j.bpc.2016.11.014. Epub 2016 Dec 3. Biophys Chem. 2017. PMID: 27939387 Free PMC article. Review.
Jackson J, Vindigni A. Jackson J, et al. Methods Mol Biol. 2022;2444:81-103. doi: 10.1007/978-1-0716-2063-2_6. Methods Mol Biol. 2022. PMID: 35290633 Free PMC article.
Quinet A, Martins DJ, Vessoni AT, Biard D, Sarasin A, Stary A, Menck CF. Quinet A, et al. Nucleic Acids Res. 2016 Jul 8;44(12):5717-31. doi: 10.1093/nar/gkw280. Epub 2016 Apr 19. Nucleic Acids Res. 2016. PMID: 27095204 Free PMC article.
Sabatinos SA, Forsburg SL. Sabatinos SA, et al. Biomolecules. 2015 Sep 18;5(3):2123-39. doi: 10.3390/biom5032123. Biomolecules. 2015. PMID: 26393661 Free PMC article. Review.
Guffanti F, Mengoli I, Damia G. Guffanti F, et al. Front Oncol. 2024 Aug 16;14:1405361. doi: 10.3389/fonc.2024.1405361. eCollection 2024. Front Oncol. 2024. PMID: 39220639 Free PMC article. Review.
Ramakrishnan N, Weaver TM, Aubuchon LN, Woldegerima A, Just T, Song K, Vindigni A, Freudenthal BD, Verma P. Ramakrishnan N, et al. Nat Commun. 2024 Jul 27;15(1):6343. doi: 10.1038/s41467-024-50673-7. Nat Commun. 2024. PMID: 39068174 Free PMC article.
Khatib JB, Dhoonmoon A, Moldovan GL, Nicolae CM. Khatib JB, et al. Nat Commun. 2024 Jul 23;15(1):6197. doi: 10.1038/s41467-024-50429-3. Nat Commun. 2024. PMID: 39043663 Free PMC article.
Fingerman DF, O'Leary DR, Hansen AR, Tran T, Harris BR, DeWeerd RA, Hayer KE, Fan J, Chen E, Tennakoon M, Meroni A, Szeto JH, Devenport J, LaVigne D, Weitzman MD, Shalem O, Bednarski J, Vindigni A, Zhao X, Green AM. Fingerman DF, et al. EMBO J. 2024 Aug;43(15):3240-3255. doi: 10.1038/s44318-024-00137-x. Epub 2024 Jun 17. EMBO J. 2024. PMID: 38886582 Free PMC article.
Kong L, Cheng C, Cheruiyot A, Yuan J, Yang Y, Hwang S, Foust D, Tsao N, Wilkerson E, Mosammaparast N, Major MB, Piston DW, Li S, You Z. Kong L, et al. Nat Commun. 2024 May 30;15(1):4609. doi: 10.1038/s41467-024-48988-6. Nat Commun. 2024. PMID: 38816425 Free PMC article.