The work in C.F.M.M. laboratory is supported by Funda&#231;&#227;o de Amparo &#224; Pesquisa do Estado de S&#227;o Paulo (FAPESP, S&#227;o Paulo, Brazil, Grants #2019/19435-3, #2013/08028-1 and 2017/05680-0) under the International Collaboration Research from FAPESP and The Netherlands Organization for Scientific Research (NWO, The Netherlands); Conselho Nacional de Desenvolvimento Cient&#237;fico e Tecnol&#243;gico (CNPq, Bras&#237;lia, DF, Brazil, Grants # 308868/2018-8] and Coordena&#231;&#227;o de Aperfei&#231;oamento de Pessoal do Ensino Superior (CAPES, Bras&#237;lia, DF, Brazil, Finance Code 001).

As the study uses human cells, the work was approved by the Ethics Committee of the Institute of Biomedical Science at the University of S&#227;o Paulo (ICB-USP, approval number #48347515.3.00005467) for the research with human samples.
NOTE: The protocols described here were used in previous publications with minor modifications14,16,28. Here the focus is on the use of ultraviolet light C (UVC) as a ...

<ol>
	<li>Rupp, W. D., Howard-Flanders, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discontinuities+in+the+DNA+synthesized+in+an+excision-defective+strain+of+Escherichia+coli+following+ultraviolet+irradiation.">Discontinuities in the DNA synthesized in an excision-defective strain of Escherichia coli following ultraviolet irradiation.</a> Journal of Molecular Biology. 31 (2), 291-304 (1968).</li><li>Meneghini, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gaps+in+DNA+synthesized+by+ultraviolet+light-irradiated+WI38+human+cells.">Gaps in DNA synthesized by ultraviolet light-irradiated WI38 human cells.</a> Biochimica et Biophysica Acta. 425 (4), 419-427 (1976).</li><li>Meneghini, R., Cordeiro-Stone, M., Schumacher, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size+and+frequency+of+gaps+in+newly+synthesized+DNA+of+xeroderma+pigmentosum+human+cells+irradiated+with+ultraviolet+light.">Size and frequency of gaps in newly synthesized DNA of xeroderma pigmentosum human cells irradiated with ultraviolet light.</a> Biophysical Journal. 33 (1), 81-92 (1981).</li><li>Lopes, M., Foiani, M., Sogo, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+mechanisms+control+chromosome+integrity+after+replication+fork+uncoupling+and+restart+at+irreparable+UV+lesions.">Multiple mechanisms control chromosome integrity after replication fork uncoupling and restart at irreparable UV lesions.</a> Molecular Cell. 21 (1), 15-27 (2006).</li><li>Heller, R. C., Marians, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+fork+reactivation+downstream+of+a+blocked+nascent+leading+strand.">Replication fork reactivation downstream of a blocked nascent leading strand.</a> Nature. 439 (7076), 557-562 (2006).</li><li>Bianchi, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PrimPol+bypasses+UV+photoproducts+during+eukaryotic+chromosomal+DNA+replication.">PrimPol bypasses UV photoproducts during eukaryotic chromosomal DNA replication.</a> Molecular Cell. 52 (4), 566-573 (2013).</li><li>Garc&#237;a-G&#243;mez, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PrimPol,+an+archaic+primase/polymerase+operating+in+human+cells.">PrimPol, an archaic primase/polymerase operating in human cells.</a> Molecular Cell. 52 (4), 541-553 (2013).</li><li>Mour&#243;n, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repriming+of+DNA+synthesis+at+stalled+replication+forks+by+human+PrimPol.">Repriming of DNA synthesis at stalled replication forks by human PrimPol.</a> Nature Structural &amp; Molecular Biology. 20 (12), 1383-1389 (2013).</li><li>Wan, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=hPrimpol1/CCDC111+is+a+human+DNA+primase-polymerase+required+for+the+maintenance+of+genome+integrity.">hPrimpol1/CCDC111 is a human DNA primase-polymerase required for the maintenance of genome integrity.</a> EMBO Reports. 14 (12), 1104-1112 (2013).</li><li>Keen, B. A., Jozwiakowski, S. K., Bailey, L. J., Bianchi, J., Doherty, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+dissection+of+the+domain+architecture+and+catalytic+activities+of+human+PrimPol.">Molecular dissection of the domain architecture and catalytic activities of human PrimPol.</a> Nucleic Acids Research. 42 (9), 5830-5845 (2014).</li><li>Tirman, S., Cybulla, E., Quinet, A., Meroni, A., Vindigni, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PRIMPOL+ready,+set,+reprime.">PRIMPOL ready, set, reprime.</a> Critical Reviews in Biochemistry and Molecular Biology. 56 (1), 17-30 (2021).</li><li>Quinet, A., Lerner, L. K., Martins, D. J., Menck, C. F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filling+gaps+in+translesion+DNA+synthesis+in+human+cells.">Filling gaps in translesion DNA synthesis in human cells.</a> Mutation Research, Genetic Toxicology Environmental Mutagenesis. 836, 127-142 (2018).</li><li>Ziv, O., Diamant, N., Shachar, S., Hendel, A., Livneh, Z., Bjergb&#230;k, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+measurement+of+translesion+DNA+synthesis+in+mammalian+cells.">Quantitative measurement of translesion DNA synthesis in mammalian cells.</a> DNA Repair Protocols. Methods in Molecular Biology (Methods and Protocols). 920, (2012).</li><li>Quinet, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translesion+synthesis+mechanisms+depend+on+the+nature+of+DNA+damage+in+UV-irradiated+human+cells.">Translesion synthesis mechanisms depend on the nature of DNA damage in UV-irradiated human cells.</a> Nucleic Acids Research. 44 (12), 5717-5731 (2016).</li><li>T&#233;cher, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+dynamics:+biases+and+robustness+of+DNA+fiber+analysis.">Replication dynamics: biases and robustness of DNA fiber analysis.</a> Journal of Molecular Biology. 425 (23), 4845-4855 (2013).</li><li>Quinet, A., Carvajal-Maldonado, D., Lemacon, D., Vindigni, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+fiber+analysis:+Mind+the+gap.">DNA fiber analysis: Mind the gap.</a> Methods in Enzymology. 591, 55-82 (2017).</li><li>Elvers, I., Johansson, F., Groth, P., Erixon, K., Helleday, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UV+stalled+replication+forks+restart+by+re-priming+in+human+fibroblasts.">UV stalled replication forks restart by re-priming in human fibroblasts.</a> Nucleic Acids Research. 39 (16), 7049-7057 (2011).</li><li>Diamant, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+damage+bypass+operates+in+the+S+and+G2+phases+of+the+cell+cycle+and+exhibits+differential+mutagenicity.">DNA damage bypass operates in the S and G2 phases of the cell cycle and exhibits differential mutagenicity.</a> Nucleic Acids Research. 40 (1), 170-180 (2012).</li><li>Temviriyanukul, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporally+distinct+translesion+synthesis+pathways+for+ultraviolet+light-induced+photoproducts+in+the+mammalian+genome.">Temporally distinct translesion synthesis pathways for ultraviolet light-induced photoproducts in the mammalian genome.</a> DNA Repair. 11 (6), 550-558 (2012).</li><li>Jansen, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redundancy+of+mammalian+Y+family+DNA+polymerases+in+cellular+responses+to+genomic+DNA+lesions+induced+by+ultraviolet+light.">Redundancy of mammalian Y family DNA polymerases in cellular responses to genomic DNA lesions induced by ultraviolet light.</a> Nucleic Acids Research. 42 (17), 11071 (2014).</li><li>Quinet, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gap-filling+and+bypass+at+the+replication+fork+are+both+active+mechanisms+for+tolerance+of+low-dose+ultraviolet-induced+DNA+damage+in+the+human+genome.">Gap-filling and bypass at the replication fork are both active mechanisms for tolerance of low-dose ultraviolet-induced DNA damage in the human genome.</a> DNA Repair. 14 (1), 27-38 (2014).</li><li>Callegari, A. J., Clark, E., Pneuman, A., Kelly, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postreplication+gaps+at+UV+lesions+are+signals+for+checkpoint+activation.">Postreplication gaps at UV lesions are signals for checkpoint activation.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (18), 8219-8224 (2010).</li><li>Vogt, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+further+properties+of+single-strand-specific+nuclease+from+Aspergillus+oryzae.">Purification and further properties of single-strand-specific nuclease from Aspergillus oryzae.</a> European Journal of Biochemistry. 33 (1), 192-200 (1973).</li><li>Cordeiro-Stone, M., Schumacher, R. I., Meneghini, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+replication+fork+in+ultraviolet+light-irradiated+human+cells.">Structure of the replication fork in ultraviolet light-irradiated human cells.</a> Biophysical Journal. 27 (2), 287-300 (1979).</li><li>Lehmann, A. R., Fuchs, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gaps+and+forks+in+DNA+replication:+Rediscovering+old+models.">Gaps and forks in DNA replication: Rediscovering old models.</a> DNA Repair (Amst). 5 (12), 1495-1498 (2006).</li><li>Daigaku, Y., Davies, A. A., Ulrich, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquitin-dependent+DNA+damage+bypass+is+separable+from+genome+replication.">Ubiquitin-dependent DNA damage bypass is separable from genome replication.</a> Nature. 465 (7300), 951-955 (2010).</li><li>Karras, G. I., Jentsch, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RAD6+DNA+damage+tolerance+pathway+operates+uncoupled+from+the+replication+fork+and+is+functional+beyond+S+phase.">The RAD6 DNA damage tolerance pathway operates uncoupled from the replication fork and is functional beyond S phase.</a> Cell. 141 (2), 255-267 (2010).</li><li>Tirman, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporally+distinct+post-replicative+repair+mechanisms+fill+PRIMPOL-dependent+ssDNA+gaps+in+human+cells.">Temporally distinct post-replicative repair mechanisms fill PRIMPOL-dependent ssDNA gaps in human cells.</a> Molecular Cell. 81 (19), 4026-4040 (2021).</li><li>Cong, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+gaps+are+a+key+determinant+of+PARP+inhibitor+synthetic+lethality+with+BRCA+deficiency.">Replication gaps are a key determinant of PARP inhibitor synthetic lethality with BRCA deficiency.</a> Molecular Cell. 81 (15), 3128-3144 (2021).</li><li>Quinet, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PRIMPOL-mediated+adaptive+response+suppresses+replication+fork+reversal+in+BRCA-deficient+cells.">PRIMPOL-mediated adaptive response suppresses replication fork reversal in BRCA-deficient cells.</a> Molecular Cell. 77 (3), 461-474 (2020).</li><li>Jackson, D. A., Pombo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replicon+clusters+are+stable+units+of+chromosome+structure:+evidence+that+nuclear+organization+contributes+to+the+efficient+activation+and+propagation+of+S+phase+in+human+cells.">Replicon clusters are stable units of chromosome structure: evidence that nuclear organization contributes to the efficient activation and propagation of S phase in human cells.</a> The Journal of Cell Biology. 140 (6), 1285-1295 (1998).</li><li>Schwab, R. A., Niedzwiedz, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+DNA+replication+in+the+vertebrate+model+system+DT40+using+the+DNA+fiber+technique.">Visualization of DNA replication in the vertebrate model system DT40 using the DNA fiber technique.</a> Journal of Visualized Experiments: JoVE. (56), e3255 (2011).</li><li>Simoneau, A., Xiong, R., Zou, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+trans+cell+cycle+effects+of+PARP+inhibitors+underlie+their+selectivity+toward+BRCA1/2-deficient+cells.">The trans cell cycle effects of PARP inhibitors underlie their selectivity toward BRCA1/2-deficient cells.</a> Genes &amp; Development. 35 (17-18), 1271-1289 (2021).</li><li>Taglialatela, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=REV1-Pol&#950;+maintains+the+viability+of+homologous+recombination-deficient+cancer+cells+through+mutagenic+repair+of+PRIMPOL-dependent+ssDNA+gaps.">REV1-Pol&#950; maintains the viability of homologous recombination-deficient cancer cells through mutagenic repair of PRIMPOL-dependent ssDNA gaps.</a> Molecular Cell. 81 (19), 4008-4025 (2021).</li><li>Lim, K. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=USP1+Is+required+for+replication+fork+protection+in+BRCA1-deficient+tumors.">USP1 Is required for replication fork protection in BRCA1-deficient tumors.</a> Molecular Cell. 72 (6), 925-941 (2018).</li><li>Peng, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opposing+roles+of+FANCJ+and+HLTF+protect+forks+and+restrain+replication+during+stress.">Opposing roles of FANCJ and HLTF protect forks and restrain replication during stress.</a> Cell Reports. 24 (12), 3251-3261 (2018).</li><li>Huang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+DNA+translocase+FANCM/MHF+promotes+replication+traverse+of+DNA+interstrand+crosslinks.">The DNA translocase FANCM/MHF promotes replication traverse of DNA interstrand crosslinks.</a> Molecular Cell. 52 (3), 434-446 (2013).</li><li>Ding, W., Bishop, M. E., Lyn-Cook, L. E., Davis, K. J., Manjanatha, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+alkaline+comet+assay+and+enzyme-modified+alkaline+comet+assay+for+measuring+DNA+strand+breaks+and+oxidative+DNA+damage+in+rat+liver.">In vivo alkaline comet assay and enzyme-modified alkaline comet assay for measuring DNA strand breaks and oxidative DNA damage in rat liver.</a> Journal of Visualized Experiments: JoVE. (111), e53833 (2016).</li><li>Lu, Y., Liu, Y., Yang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+in+vitro+DNA+damage+using+comet+assay.">Evaluating in vitro DNA damage using comet assay.</a> Journal of Visualized Experiments: JoVE. (128), e56450 (2017).</li></ol>

The authors have nothing to disclose.

Critical steps of the standard DNA fiber assay protocol were discussed in a previous publication32. Here, we describe modified versions of the standard DNA fiber assay to investigate the presence of post-replicative ssDNA gaps as well as their repair by gap-filling, initially described in14. In the context of post-replicative ssDNA gap presence, the use of the S1 nuclease in the S1 Fiber protocol would most likely be suitable after exposure of a genotoxic agent for a minimu...

Seminal works have provided evidence of the accumulation of post-replicative single-stranded (ssDNA) gaps upon treatment with DNA damaging agents in bacteria1 and human cells2,3. During replication of damaged DNA templates, the DNA synthesis machinery may bypass the lesions by employing specific translesion synthesis DNA polymerases or through template switching mechanisms. Alternatively, the replisome may also simply skip the lesion leaving an ssDNA gap behind, to be repaired later. More recently, a study clearly showed that treatment with genotoxic agents leads to ssDNA gaps in eukaryotes by utilizing electron microscopy to visualize the specific structure of replication intermediates4. The formation of these regions of post-replicative gaps was initially proposed to be a simple result of the semi-discontinuous mode of DNA replication2. In this case, a lesion on the lagging strand can block the elongation of an Okazaki fragment, but fork progression is naturally rescued by the following Okazaki fragment, leaving behind an ssDNA gap. However, further studies demonstrated that the formation of gaps on the leading strand is also possible, as first shown in bacteria5. In eukaryotes, PRIMPOL, a unique DNA polymerase with primase activity, was shown to be able to restart DNA synthesis downstream a replication blocking lesion through its repriming activity6,7,8,9,10,11. Thus, the PRIMPOL primase activity may explain the formation of post-replicative ssDNA gaps in the leading strand upon treatment with a DNA damaging agent in human cells12. Nonetheless, detection of these gaps as well as gap repair, until recently, required indirect or time-consuming approaches such as electron microscopy4 or plasmid-based assays13. The use of the ssDNA-specific S1 nuclease to detect gaps in human cells was pioneered by early studies more than forty years ago, using sucrose gradient techniques2,3. More recently, our group applied the use of this nuclease to detect ssDNA in replicating DNA (post-replicative gaps) using other methods such as DNA fiber and comet assays14. These new approaches paved the way for the current surge of studies on post-replicative gaps. Here, we describe a strategy of using S1 nuclease to detect post-replicative ssDNA gaps by DNA fiber assay and explain how a differential labeling scheme in the DNA fiber protocol can permit the study of the repair of these gaps.
The DNA fiber assay is a powerful technique that has been used by a growing number of labs and has provided valuable insights into replication fork dynamics and replication stress response mechanisms. Briefly, this technique is based on the sequential incorporation of nucleotide analogs (such as CldU -5-chloro-2&#39;-deoxyuridine-, and IdU -5-iodo-2&#39;-deoxyuridine) in the replicating DNA. After harvesting, cells are lysed, and DNA molecules spread on a positively coated glass slide. CldU and IdU are then detected by specific antibodies, which can be visualized in a fluorescent microscope as bicolored fibers. Finally, the lengths of the IdU and CldU tracts are measured to identify any alterations of DNA replication dynamics as a consequence of DNA damage induction. This technique can be utilized to investigate different phenomena, such as fork stalling, fork slowing, nascent DNA degradation, and variations in the frequency of origin firing15,16.
One limitation of the DNA fiber assay is its resolution of a few kilobases. Because post-replicative ssDNA gaps can be in the range of hundreds of bases, it is impossible to visualize these gaps directly by the standard DNA fiber protocol. The presence of ssDNA gaps on replicating DNA in human cells treated with genotoxic agents has been indirectly implicated before. For example, the observation of ssDNA as assessed by recruitment of the ssDNA-binding protein, replication protein A (RPA), in cells outside the S phase, or the formation of ssDNA as detected by alkaline DNA unwinding combined with the absence of prolonged fork stalling by DNA fiber assay12,17,18,19,20,21 was attributed to the accumulation of ssDNA gaps. In addition, post-replicative ssDNA gaps induce an ATR-dependent G2/M phase checkpoint and arrest cells treated with replication poisons18,19,20,21,22.
The S1 nuclease degrades single-stranded nucleic acids releasing 5&#39;-phosphoryl mono- or oligonucleotides and has a 5-times higher affinity to ssDNA compared to RNA. Double-stranded nucleic acids (DNA:DNA, DNA:RNA, or RNA:RNA) are resistant to the S1 nuclease except when used in extremely high concentrations. The S1 nuclease also cleaves double-stranded DNA at the single-stranded region caused by a nick, gap, mismatch, or loop. The S1 nuclease is thus an ssDNA-specific endonuclease capable of cleaving ssDNA gaps, ultimately generating double-stranded breaks23,24. Thus, adding steps for S1 nuclease digestion to the DNA fiber protocol indirectly enables the detection of post-replicative ssDNA gaps. In the presence of gaps, treatment of exposed nuclei with the S1 nuclease prior to DNA spreading will generate shorter tracts as a consequence of S1 cleavage of ssDNA inherently present at gaps14. Accordingly, tract shortening is the read-out for ssDNA gaps using this approach. Compared to the standard DNA fiber protocol, the DNA fiber with the S1 nuclease only requires two extra steps: nuclei exposure (cell permeabilization) and treatment with the S1 nuclease. It is important to note that appropriate controls are mandatory, such as samples treated with the genotoxic agent but without the S1 nuclease and samples treated with the S1 nuclease without the genotoxic agent. The protocol itself, including the incorporation of analogs, S1 treatment, and spreading, can be performed in one day and does not require exceptional material. It only requires the thymidine analogs, the purified S1 nuclease, the appropriate primary and secondary antibodies, and a fluorescent microscope. Overall, the DNA fiber employing the S1 nuclease detects ssDNA gaps on ongoing replication forks using a relatively simple approach.
The post-replicative ssDNA gaps formed as a consequence of replication stress response mechanisms can be repaired (or filled) by different mechanisms, including translesion DNA synthesis or template switching, in a process called gap-filling or post-replication repair (PRR)25. These processes occur behind the advancing forks, involving replication-independent DNA synthesis14,26,27. Based on these findings, a labeling scheme distinct from the standard DNA fiber assay can be performed to visualize gap-filling events in the G2 phase directly14,16,26,28. Specifically, one thymidine analog can be used to label the replication fork at the time of genotoxic treatment and post-replicative ssDNA gap formation, while another thymidine analog can be used to label gap-filling events. In this protocol, cells are labeled with a first thymidine analog (IdU, for example) immediately after or concomitantly with genotoxic treatment for 1 h, so that the nascent DNA is labeled at the time of gap formation. Nocodazole is added upon treatment for anywhere between 12-24 h to arrest cells in G2/M, preventing the following S phase. For the last 4 h of nocodazole treatment, a second thymidine analog (CldU, for example) is added to the media to be incorporated during gap filling. Importantly, this assay can only be used to detect gap-filling events in G2 because the gap-filling signal from CldU cannot be distinguished from a signal due to CldU incorporation into replicating DNA in the S phase. Therefore, to minimize the background signal, the timing of CldU incorporation after damage induction should coincide with when most of the cell population is entering the G2 phase14. Therefore, this timing will vary depending on cell line and treatment conditions. Optimizing cell cycle progression prior to employing this assay is advised. Co-staining of these thymidine analogs allows the visualization of gap-filling (PRR) tracts (CldU patches) on top of nascent DNA (IdU tracts) synthesized during the genotoxic treatment when ssDNA gaps were generated.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetic acid, Glacial</td>
			<td>Synth</td>
			<td>64-19-7</td>
			<td>Alternatively, BSA - Biosera - REF PM-T1725/100</td>
		</tr>
		<tr>
			<td>Ammonium hydroxide</td>
			<td>Synth</td>
			<td>1336-21-6</td>
			<td>Or similar</td>
		</tr>
		<tr>
			<td>Antibody anti-mouse IgG1 Alexa Fluor 594</td>
			<td>Invitrogen</td>
			<td>A11005</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Antibody anti-rat Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A21470</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Antibody Mouse anti-BrdU</td>
			<td>Becton Disckson</td>
			<td>347580</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Antibody Rat anti-BrdU</td>
			<td>Abcam</td>
			<td>&#160;Ab6326</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Biological security hood</td>
			<td>Pachane</td>
			<td>PA 410</td>
			<td>Use hood present in the laboratory</td>
		</tr>
		<tr>
			<td>BSA (Bovine Serum Albumin)</td>
			<td>Sigma-Aldrich</td>
			<td>A3294</td>
			<td>Or similar</td>
		</tr>
		<tr>
			<td>Cell scraper</td>
			<td>Thermo Scientific</td>
			<td>179693</td>
			<td>Or similar</td>
		</tr>
		<tr>
			<td>CldU</td>
			<td>Millipore-Sigma</td>
			<td>C6891</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Cloridric acid</td>
			<td>Synth</td>
			<td>7647-01-0</td>
			<td>Or similar</td>
		</tr>
		<tr>
			<td>Confocal Zeiss LSM Series (7, 8 or 9)</td>
			<td>Zeiss</td>
			<td>-</td>
			<td>Or similar</td>
		</tr>
		<tr>
			<td>Cover glass (or coverslips)</td>
			<td>Thermo Scientific</td>
			<td>152460</td>
			<td>Alternatively, Olen - Kasvi Cover Glass (24 x 60 mm) - K5-2460</td>
		</tr>
		<tr>
			<td>DMEM - High Glucose</td>
			<td>LGC/Gibco</td>
			<td>BR30211-05/12100046</td>
			<td>Use culture media specific for the cell line used.</td>
		</tr>
		<tr>
			<td>EDTA (Ethylenediaminetetraacetic acid disodium salt dihydrate)</td>
			<td>Sigma-Aldrich</td>
			<td>E5314</td>
			<td>Or similar</td>
		</tr>
		<tr>
			<td>Epifluorescence Microscope Axiovert 200</td>
			<td>Zeiss</td>
			<td>-</td>
			<td>Or similar</td>
		</tr>
		<tr>
			<td>FBS (Fetal Bovine Serum)</td>
			<td>Gibco</td>
			<td>12657-029</td>
			<td>Or similar</td>
		</tr>
		<tr>
			<td>Forma Series II Water Jacketed CO2 Incubator</td>
			<td>Thermo Scientific 3110</td>
			<td>13-998-074</td>
			<td>Use cell incubator present in the laboratory</td>
		</tr>
		<tr>
			<td>Glass slide jar</td>
			<td>Sigma-Aldrich</td>
			<td>S5516</td>
			<td>Or similar</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>56-81-5</td>
			<td>Or similar</td>
		</tr>
		<tr>
			<td>Idu</td>
			<td>Millipore-Sigma</td>
			<td>I7125</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Magnesium Chloride</td>
			<td>Synth</td>
			<td>7791-18-6</td>
			<td>Or similar</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Merck</td>
			<td>67-56-1</td>
			<td>Or similar</td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Denville</td>
			<td>M1021</td>
			<td>Alternatively, Olen - Kasvi Microscope Slides - K5-7105 OR Precision Glass Line - 7105-1</td>
		</tr>
		<tr>
			<td>MOPS (&#193;cido 3-morfolinopropano 1-sulf&#244;nico)</td>
			<td>Synth</td>
			<td>1132-61-2</td>
			<td>Or similar</td>
		</tr>
		<tr>
			<td>Nocodazole</td>
			<td>Sigma-Aldrich</td>
			<td>31430-18-9</td>
			<td>-</td>
		</tr>
		<tr>
			<td>PBS (Phosphate Buffer Saline)</td>
			<td>Life Thechnologies</td>
			<td>3002</td>
			<td>Or similar</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td>Or similar</td>
		</tr>
		<tr>
			<td>ProLong Gold AntiFade Mountant</td>
			<td>Invitrogen</td>
			<td>P36930</td>
			<td>Any antifade moutant solution for immunofluorescence could be used</td>
		</tr>
		<tr>
			<td>S1 nuclease purified from Aspergillus oryzae</td>
			<td>Invitrogen</td>
			<td>18001-016</td>
			<td>Pre-dilute the S1 nuclease (1/100 - 1/200) in S1 nuclease dilution buffer provided by the manufacturer, aliquote and store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>SDS (Sodium Dodecyl Sulfate)</td>
			<td>BioRad</td>
			<td>161-0302</td>
			<td>Or similar</td>
		</tr>
		<tr>
			<td>Sodium Acetate Trihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>6131-90-4</td>
			<td>Or similar</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Synth</td>
			<td>&#160;7647-14-5</td>
			<td>Or similar</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>57-50-1</td>
			<td>Or similar</td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>West Lab Research</td>
			<td>BP152-1</td>
			<td>Or similar</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Synth</td>
			<td>9002-93-1</td>
			<td>Or similar</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Gibco</td>
			<td>25200072</td>
			<td>Or similar</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma-Aldrich</td>
			<td>P1379</td>
			<td>Or similar</td>
		</tr>
		<tr>
			<td>UVC Lamp</td>
			<td>Non Specific</td>
			<td>-</td>
			<td>Essential: emission lenght of 254 nm</td>
		</tr>
		<tr>
			<td>VLX-3W UV Radiometer</td>
			<td>Vilber Loumart</td>
			<td>-</td>
			<td>Or similar</td>
		</tr>
		<tr>
			<td>Zinc Acetate</td>
			<td>Sigma-Aldrich</td>
			<td>557-34-6</td>
			<td>Or similar</td>
		</tr>
	</tbody>
</table>

detection of post-replicative gaps accumulation and repair in human cells using the dna fiber assay

The DNA fiber assay is a simple and robust method for the analysis of replication fork dynamics, based on the immunodetection of nucleotide analogs that are incorporated during DNA synthesis in human cells. However, this technique has a limited resolution of a few thousand kilobases. Consequently, post-replicative single-stranded DNA (ssDNA) gaps as small as a few hundred bases are not detectable by the standard assay. Here, we describe a modified version of the DNA fiber assay that utilizes the S1 nuclease, an enzyme that specifically cleaves ssDNA. In the presence of post-replicative ssDNA gaps, the S1 nuclease will target and cleave the gaps, generating shorter tracts that can be used as a read-out for ssDNA gaps on ongoing forks. These post-replicative ssDNA gaps are formed when damaged DNA is replicated discontinuously. They can be repaired via mechanisms uncoupled from genome replication, in a process known as gap-filling or post-replicative repair. Because gap-filling mechanisms involve DNA synthesis independent of the S phase, alterations in the DNA fiber labeling scheme can also be employed to monitor gap-filling events. Altogether, these modifications of the DNA fiber assay are powerful strategies to understand how post-replicative gaps are formed and filled in the genome of human cells.

In the S1 Fiber assay, if treatment with a genotoxic agent leads to post-replicative ssDNA gaps, the overall lengths of DNA fibers from S1-treated nuclei will be shorter upon treatment with DNA damage compared to untreated samples as well as to samples that were treated with the genotoxic agent but were not submitted to S1 cleavage (Figure 1).
Alternatively, if treatment with the S1 nuclease does not significantly affect the length of DNA fibers compared to untrea...

Watch this Scientific Journal Video about Detection of Post-Replicative Gaps Accumulation and Repair in Human Cells Using the DNA Fiber Assay at JoVE.com

Detection of Post-Replicative Gaps Accumulation and Repair in Human Cells Using the DNA Fiber Assay

Here we describe two modifications of the DNA fiber assay to investigate single-stranded DNA gaps in replicating DNA after lesion induction. The S1 fiber assay enables the detection of post-replicative gaps using the ssDNA-specific S1 endonuclease, while the gap-filling assay allows visualization and quantification of gap repair.

The DNA fiber assay is a simple and robust method for the analysis of replication fork dynamics, based on the immunodetection of nucleotide analogs that ...

Our protocol opens new possibilities to investigate how replication occurs on damaged DNA. The techniques can detect the formation and repair of single-stranded DNA gaps, opposite the DNA lesion. The S1 nuclease cleaves the single-stranded DNA and allows the detection of post-replicative gaps.This approach was first used by Dr.Meneghini and Dr.Cordiero-Stone in the 1970s in Brazil, and recently adapted by us to the DNA fiber assay. Using the technique described here to study the formation and repair of post-replicative gaps can provide insights into the mechanism of DNA repair and replication stress response that maintain genome integrity. For both protocols described here, the S1 fiber and the gap-filling assays, It is crucial to include all the controls to ensure accurate interpretation of the data.The procedure will be demonstrated by Davi J.Martins, a PhD student from our lab, and Dr.Annabel Quinet, a collaborator who previously developed these techniques when working in our group. To begin with, aspirate the culture media and immediately add the culture media with 20-micromolar of IDU media to the cells. Incubate the cells at 37 degrees Celsius and 5%carbon dioxide for precisely 20 minutes.Then, quickly wash the cells with pre-warmed PBS twice and irradiate them with 20 joules per meter square UV-C. Use untreated cells as controls. Immediately add the media with 200-micromolar CldU to the cells and incubate them at 37 degrees Celsius and 5%carbon dioxide for precisely 60 minutes.After that, wash the cells with pre-warmed PBS twice and permeabilize them with the CSK 100 buffer for two to 10 minutes according to the cell line at room temperature. After CSK permeabilization, the nuclei with almost no cytoplasm can be seen compared to those before the permeabilization. Carefully pour PBS down the side of each well to wash the nuclei with PBS.Then, wash the nuclei with S1 buffer. Incubate the nuclei in the S1 buffer with 20 units per milliliter S1 nuclease, and without the S1 nuclease, for 30 minutes at 37 degrees Celsius. Next, aspirate the S1 buffer and add 0.1%BSA in PBS.Scrape the nuclei and transfer them to an approximately annotated 1.5-milliliter tube. Keep the tubes on ice all the time. Centrifuge the nuclei at around 4, 600 times G for five minutes at four degrees Celsius.Resuspend the pellets in PBS at a concentration of approximately 1, 500 cells per microliter. Place the tubes on ice and immediately proceed to DNA fiber preparation by spreading protocol. Annotate the microscope slides with a carbon pencil and place them flat on a tray.Tap the bottom of the tube to ensure homogenization and add two microliters of cells on the top of the corresponding slide. Add six microliters of the lysis buffer and lyse the cells by pipetting up and down about five times. Use the pipette tip to pull the drop slightly towards the bottom of the slide and incubate for five minutes at room temperature to lyse the nuclei.Tilt the slide at around 20 to 40 degrees and allow the drop to spread to the bottom of the slide at a constant low speed. Allow the slide to dry at room temperature in the dark for 10 to 15 minutes. Then, immerse them in a jar containing the freshly prepared fixing solution and incubate for five minutes at room temperature to fix DNA onto the slides.Wash the slides with PBS twice for five minutes and denature the DNA with freshly prepared 2.5-molars cloridric acid for 40 to 60 minutes at room temperature. Wash the slides with PBS three times for five minutes and block with 5%BSA previously warmed for 30 to 60 minutes at 37 degrees Celsius. Gently tap on the paper to remove the excess liquid from the slides.Add 30 microliters of primary antibodies to the slides dropwise and place a cover slip on top. Incubate for 1 to 1.5 hours at room temperature in a dark, humid chamber. Place the slides in a jar with PBS for one to two minutes and then carefully remove the cover slips.Wash with PBST in a jar three times for five minutes and place the slides in PBS. Remove the excess liquid by gently tapping on a paper and add 30 microliters of the secondary antibodies to the slides dropwise. Place a cover slip on top.Incubate for 45 to 60 minutes at room temperature in a dark and humid chamber. Repeat the steps for washing the slides and removing the excess PBS once more and add 20 microliters of the mounting reagent dropwise to the slides. Place a cover slip on top and avoid bubbles.Place a drop of immersion oil near the top of the slide where the cells were lysed. Use the green channel and search the green dots in the background to find the focus. Open the Leica Application Suite software, click on Acquire, and select the green channel.Adjust the settings of the green channel before analysis. Then, change to the red channel and adjust the settings. Find the main concentration of fibers and move to the edges to find the well spread, non-overlapping, individual fibers.Use only one channel to select the regions for the pictures and avoid potential bias. Click on the Acquire Overlay icon and adjust the software for the correct microscope magnification. Take around 15 to 20 pictures per slide alongside the entire slide at the green channel and then at the red channel.The two channels are now merged and the created overlay is moved to a unique folder. It is possible to see the fibers with red and green colors. Open the ImageJ software, left click on the fifth icon, select the second option, and then select the Segmented Line tool.Left click to draw the exact path of the red or green tract and right click to stop the drawing. Push the M button on the keyboard to measure the length. For the S1 fiber, keep track of the red and green tracts measurements for each fiber and measure red and green tract lengths from at least 150 fibers from different pictures.An S1 fiber is shown in these representative images. Control-nascent DNA tract without the gaps and impervious to cleavage by the S1 nuclease is shown here. DNA tract positive for gaps cleaved by the S1 nuclease resulted in a shorter CldU tract length.A differential labeling scheme extensively described in the text manuscript can be performed to label gap-filling events. In this assay, named gap-filling assay, longer IDU tracks with at least one CldU patch or fewer CldU patches correspond with low gap-filling density. Shorter IDU tracts, with at least one CldU patch, and longer IDU tracts with more CldU patches, correspond with high gap-filling density.The permeabilization step of this protocol is crucial for the S1 nuclease to act on the gaps formed during the replication of damaged DNA. The S1 nuclease can also be used in the comet assay protocol to detect the gaps formed during the replication of damaged DNA that persists after treatment. The S1 fiber assay paved the way for the new paradigm that post-replicative gaps can cause cancer vulnerability, particularly in breast and ovarian cancers carrying mutations in BRCA1 and 2 genes.

detection-post-replicative-gaps-accumulation-repair-human-cells-using

Research

JoVE Journal

Genetics

5.3K visualizações.  University of São Paulo. Nosso protocolo abre novas possibilidades para investigar como a replicação ocorre no DNA danificado. As técnicas podem detectar a formação e reparação de lacunas de DNA de um único fio, em frente à lesão de DNA. A nuclease S1 corta o DNA de uma única fenda e permite a detecção de lacunas pós-replicativas.Essa abordagem foi usada pela primeira vez pelo Dr.Meneghini e pelo Dr.Cordiero-Stone na década de 1970 no Brasil, e recentemente adaptada por nós ao ensaio de fibra d...