We have developed a simple system for the analysis of the affinity of anti-bromodeoxyuridine antibodies. The system is based on the anchored oligonucleotides containing 5-bromo-2'-deoxyuridine (BrdU) at three different positions. It allows a reliable estimation of the reactivity of particular clones of monoclonal anti-bromodeoxyuridine antibodies with BrdU in fixed and permeabilized cells. Using oligonucleotide probes and four different protocols for the detection of BrdU incorporated in cellular DNA, we identified two antibody clones that evinced sufficient reactivity to BrdU in all the tested protocols. One of these clones exhibited higher reactivity to 5-iodo-2'-deoxyuridine (IdU) than to BrdU. It allowed us to increase the sensitivity of the used protocols without a negative effect on the cell physiology as the cytotoxicity of IdU was comparable with BrdU and negligible when compared to 5-ethynyl-2'-deoxyuridine. The combination of IdU and the improved protocol for oxidative degradation of DNA provided a sensitive and reliable approach for the situations when the low degradation of DNA and high BrdU signal is a priority.

Institute of Molecular and Translational Medicine Faculty of Medicine Palacký University Olomouc Czech Republic

Oligonucleotide group Institute of Organic Chemistry and Biochemistry CAS v v i Prague Czech Republic

Zobrazit více v PubMed

Ageno M, Dore E, Frontali C. The alkaline denaturation of DNA. Biophys J. 1969; 9: 1281–1311. PubMed PMC

Dimitrova DS, Berezney R. The spatio-temporal organization of DNA replication sites is identical in primary, immortalized and transformed mammalian cells. J Cell Sci. 2002; 115: 4037–4051. PubMed

Jackson DA, Pombo A. 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. J Cell Biol. 1998; 140: 1285–1295. PubMed PMC

Kennedy BK, Barbie DA, Classon M, Dyson N, Harlow E. Nuclear organization of DNA replication in primary mammalian cells. Genes Dev. 2000; 14: 2855–2868. PubMed PMC

Ligasová A, Strunin D, Liboska R, Rosenberg I, Koberna K. Atomic scissors: a new method of tracking the 5-bromo-2'-deoxyuridine-labeled DNA in situ. PLoS One. 2012; 7: e52584 10.1371/journal.pone.0052584 PubMed DOI PMC

Stanojcic S, Lemaitre JM, Brodolin K, Danis E, Mechali M. In Xenopus egg extracts, DNA replication initiates preferentially at or near asymmetric AT sequences. Mol Cell Biol. 2008; 28: 5265–5274. 10.1128/MCB.00181-08 PubMed DOI PMC

Tang X, Falls DL, Li X, Lane T, Luskin MB. Antigen-retrieval procedure for bromodeoxyuridine immunolabeling with concurrent labeling of nuclear DNA and antigens damaged by HCl pretreatment. J Neurosci. 2007; 27: 5837–5844. PubMed PMC

Salic A, Mitchison TJ. A chemical method for fast and sensitive detection of DNA synthesis in vivo. Proc Natl Acad Sci U S A. 2008; 105: 2415–2420. 10.1073/pnas.0712168105 PubMed DOI PMC

Hong V, Steinmetz NF, Manchester M, Finn MG. Labeling live cells by copper-catalyzed alkyne—azide click chemistry. Bioconjug Chem. 2010; 21: 1912–1916. 10.1021/bc100272z PubMed DOI PMC

Loschberger A, Niehorster T, Sauer M. Click chemistry for the conservation of cellular structures and fluorescent proteins: ClickOx. Biotechnol J. 2014; 9: 693–697. 10.1002/biot.201400026 PubMed DOI

Ligasová A, Strunin D, Friedecký D, Adam T, Koberna K. A fatal combination: a thymidylate synthase inhibitor with DNA damaging activity. PLoS One. 2015; 10: e0117459 10.1371/journal.pone.0117459 PubMed DOI PMC

Cristofoli WA, Wiebe LI, De Clercq E, Andrei G, Snoeck R, Balzarini J, et al. 5-alkynyl analogs of arabinouridine and 2'-deoxyuridine: cytostatic activity against herpes simplex virus and varicella-zoster thymidine kinase gene-transfected cells. J Med Chem. 2007; 50: 2851–2857. PubMed

Diermeier-Daucher S, Clarke ST, Hill D, Vollmann-Zwerenz A, Bradford JA, Brockhoff G. Cell type specific applicability of 5-ethynyl-2'-deoxyuridine (EdU) for dynamic proliferation assessment in flow cytometry. Cytometry A. 2009; 75: 535–546. 10.1002/cyto.a.20712 PubMed DOI

Kohlmeier F, Maya-Mendoza A, Jackson DA. EdU induces DNA damage response and cell death in mESC in culture. Chromosome Res. 2013; 21: 87–100. 10.1007/s10577-013-9340-5 PubMed DOI PMC

Meneni S, Ott I, Sergeant CD, Sniady A, Gust R, Dembinski R. 5-Alkynyl-2'-deoxyuridines: chromatography-free synthesis and cytotoxicity evaluation against human breast cancer cells. Bioorg Med Chem. 2007; 15: 3082–3088. PubMed PMC

Ross HH, Rahman M, Levkoff LH, Millette S, Martin-Carreras T, Dunbar EM, et al. Ethynyldeoxyuridine (EdU) suppresses in vitro population expansion and in vivo tumor progression of human glioblastoma cells. J Neurooncol. 2011; 105: 485–498. 10.1007/s11060-011-0621-6 PubMed DOI PMC

Zhao H, Halicka HD, Li J, Biela E, Berniak K, Dobrucki J, et al. DNA damage signaling, impairment of cell cycle progression, and apoptosis triggered by 5-ethynyl-2'-deoxyuridine incorporated into DNA. Cytometry A. 2013; 83: 979–988. 10.1002/cyto.a.22396 PubMed DOI PMC

Pepperkok R, Ansorge W. Direct visualization of DNA replication sites in living cells by microinjection of fluorescein conjugated dUTPs. Methods Mol Cell Biol. 1995; 5: 112–117.

Koberna K, Stanek D, Malinsky J, Eltsov M, Pliss A, Ctrnacta V, et al. Nuclear organization studied with the help of a hypotonic shift: its use permits hydrophilic molecules to enter into living cells. Chromosoma. 1999; 108: 325–335. PubMed

Stanek D, Koberna K, Pliss A, Malinsky J, Masata M, Vecerova J, et al. Non-isotopic mapping of ribosomal RNA synthesis and processing in the nucleolus. Chromosoma. 2001; 110: 460–470. PubMed

Ligasová A, Raska I, Koberna K. Organization of human replicon: singles or zipping couples? J Struct Biol. 2009; 165: 204–213. 10.1016/j.jsb.2008.11.004 PubMed DOI PMC

Liboska R, Ligasova A, Strunin D, Rosenberg I, Koberna K. Most anti-BrdU antibodies react with 2'-deoxy-5-ethynyluridine—the method for the effective suppression of this cross-reactivity. PLoS One. 2012; 7: e51679 10.1371/journal.pone.0051679 PubMed DOI PMC

Sporbert A, Gahl A, Ankerhold R, Leonhardt H, Cardoso MC. DNA polymerase clamp shows little turnover at established replication sites but sequential de novo assembly at adjacent origin clusters. Mol Cell. 2002; 10: 1355–1365. PubMed

Freshney RI. Cytotoxicity In: Freshney RI, editor. Culture of Animal Cells A manual of basic techniques. 5th. ed. New Jersey: John Wiley and Sons, Inc; 2005. pp 365–369.

Ahmad-Zadeh C, Piguet JD, Colli L. Molecular weight estimation of immunoglobulin subunits of polyacrylamide gel. Immunology. 1971; 21: 1065–1071. PubMed PMC

Aten JA, Bakker PJ, Stap J, Boschman GA, Veenhof CH. DNA double labelling with IdUrd and CldUrd for spatial and temporal analysis of cell proliferation and DNA replication. Histochem J. 1992; 24: 251–259. PubMed

Zink D, Cremer T, Saffrich R, Fischer R, Trendelenburg MF, Ansorge W, et al. Structure and dynamics of human interphase chromosome territories in vivo. Hum Genet. 1998; 102: 241–251. PubMed

Dimitrova DS, Gilbert DM. The spatial position and replication timing of chromosomal domains are both established in early G1 phase. Mol Cell. 1999; 4: 983–993. PubMed

Dimitrova DS, Todorov IT, Melendy T, Gilbert DM. Mcm2, but not RPA, is a component of the mammalian early G1-phase prereplication complex. J Cell Biol. 1999; 146: 709–722. PubMed PMC

Sokol AM, Cruet-Hennequart S, Pasero P, Carty MP. DNA polymerase eta modulates replication fork progression and DNA damage responses in platinum-treated human cells. Sci Rep. 2013; 3: 3277 10.1038/srep03277 PubMed DOI PMC

Campbell VW, Jackson DA. The effect of divalent cations on the mode of action of DNase I. The initial reaction products produced from covalently closed circular DNA. J Biol Chem. 1980; 255: 3726–3735. PubMed

Junowicz E, Spencer JH. Studies on bovine pancreatic deoxyribonuclease A. II. The effect of different bivalent metals on the specificity of degradation of DNA. Biochim Biophys Acta. 1973; 312: 85–102. PubMed

Junowicz E, Spencer JH. Studies on bovine pancreatic deoxyribonuclease A. I. General properties and activation with different bivalent metals. Biochim Biophys Acta. 1973; 312: 72–84. PubMed

Ligasova A, Strunin D, Koberna K. A New Method of the Visualization of the Double-Stranded Mitochondrial and Nuclear DNA. PLoS One. 2013; 8: e66864 PubMed PMC

Barbie DA, Kudlow BA, Frock R, Zhao J, Johnson BR, Dyson N, et al. Nuclear reorganization of mammalian DNA synthesis prior to cell cycle exit. Mol Cell Biol. 2004; 24: 595–607. PubMed PMC

The kinetics of uracil-N-glycosylase distribution inside replication foci

New Concept and Apparatus for Cytocentrifugation and Cell Processing for Microscopy Analysis

DNA Replication: From Radioisotopes to Click Chemistry

Cell cycle profiling by image and flow cytometry: The optimised protocol for the detection of replicational activity using 5-Bromo-2'-deoxyuridine, low concentration of hydrochloric acid and exonuclease III

Looking for ugly ducklings: The role of the stability of BrdU-antibody complex and the improved method of the detection of DNA replication

Dr Jekyll and Mr Hyde: a strange case of 5-ethynyl-2'-deoxyuridine and 5-ethynyl-2'-deoxycytidine