Abstract
The misreplication of damaged DNA, a biological process termed translesion DNA synthesis (TLS), produces a large number of adverse effects on human health. This chapter describes the application of an artificial nucleoside/nucleotide system that functions as a biochemical probe to quantify TLS activity under in vitro and in vivo conditions. For in vitro studies, the artificial nucleotide, 3-ethynyl-5-nitroindolyl-2′-deoxyriboside triphosphate (3-Eth-5-NITP), is used as it is efficiently inserted opposite an abasic site, a highly pro-mutagenic DNA lesion produced by several types of DNA-damaging agents. The placement of the ethynyl moiety allows the incorporated nucleoside triphosphate to be selectively tagged with azide-containing fluorophores via “click” chemistry. This reaction provides a facile way to quantify the extent of nucleotide incorporation opposite this and other noninstructional DNA lesions. The corresponding nucleoside, 3-Eth-5-NIdR, can be used to monitor TLS activity in hematological and adherent cancer cells treated with compounds that produce noninstructional DNA lesions. As described above, visualizing the replication of these lesions is achieved using copper-catalyzed “click” chemistry to tag the ethynyl moiety present on the nucleotide with fluorogenic probes. This technique represents a new diagnostic approach to quantify TLS activity inside cells. In addition, the application of this “clickable” nucleoside provides a chemical probe to identify cells that become drug resistant by the facile replication of noninstructional DNA lesions produced by DNA-damaging agents.
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References
Greenberg MM (2012) The formamidopyrimidines: purine lesions formed in competition with 8-oxopurines from oxidative stress. Acc Chem Res 45:588–597
Cadet J, Douki T, Ravanat JL (2008) Oxidatively generated damage to the guanine moiety of DNA: mechanistic aspects and formation in cells. Acc Chem Res 41:1075–1083
Neeley WL, Essigmann JM (2006) Mechanisms of formation, genotoxicity, and mutation of guanine oxidation products. Chem Res Toxicol 19:491–505
Krokan HE, Bjørås M (2013) Base excision repair. Cold Spring Harb Perspect Biol 5:a012583
Spivak G (2015) Nucleotide excision repair in humans. DNA Repair (Amst) 36:13–18
Jasin M, Rothstein R (2013) Repair of strand breaks by homologous recombination. Cold Spring Harb Perspect Biol 5:a012740
Goodman MF, Tippin B (2000) Sloppier copier DNA polymerases involved in genome repair. Curr Opin Genet Dev 10:162–168
Goodman MF, Woodgate R (2013) Translesion DNA polymerases. Cold Spring Harb Perspect Biol 5:a010363
Sale JE (2013) Translesion DNA synthesis and mutagenesis in eukaryotes. Cold Spring Harb Perspect Biol 5:a012708
Sutton MD, Walker GC (2001) Managing DNA polymerases: coordinating DNA replication, DNA repair, and DNA recombination. Proc Natl Acad Sci U S A 98:8342–8349
Zhao L, Washington MT (2017) Translesion synthesis: insights into the selection and switching of DNA polymerases. Genes (Basel) 8(1). pii: E24
Zhao J, Yu S, Zheng Y, Yang H, Zhang J (2017) Oxidative modification and its implications for the neurodegeneration of Parkinson’s disease. Mol Neurobiol 54:1404–1418
Makridakis NM, Reichardt JK (2012) Translesion DNA polymerases and cancer. Front Genet 3:174
Kunz BA, Straffon AF, Vonarx EJ (2000) DNA damage-induced mutation: tolerance via translesion synthesis. Mutat Res 451:169–185
Calderón-Montaño JM, Burgos-Morón E, Orta ML, López-Lázaro M (2014) Effect of DNA repair deficiencies on the cytotoxicity of drugs used in cancer therapy – a review. Curr Med Chem 21:3419–3454
Nicolay NH, Helleday T, Sharma RA (2012) Biological relevance of DNA polymerase β and translesion synthesis polymerases to cancer and its treatment. Curr Mol Pharmacol 5:54–67
Roy U, Schärer OD (2016) Involvement of translesion synthesis DNA polymerases in DNA interstrand crosslink repair. DNA Repair (Amst) 44:33–41
Haynes B, Saadat N, Myung B, Shekhar MP (2015) Crosstalk between translesion synthesis, Fanconi anemia network, and homologous recombination repair pathways in interstrand DNA crosslink repair and development of chemoresistance. Mutat Res Rev Mutat Res 763:258–266
Salehan MR, Morse HR (2013) DNA damage repair and tolerance: a role in chemotherapeutic drug resistance. Br J Biomed Sci 70:31–40
Devadoss B, Lee I, Berdis AJ (2013) Spectroscopic analysis of polymerization and exonuclease proofreading by a high-fidelity DNA polymerase during translesion DNA synthesis. Biochim Biophys Acta 1834:34–45
Motea EA, Lee I, Berdis AJ (2012) A non-natural nucleoside with combined therapeutic and diagnostic activities against leukemia. ACS Chem Biol 7:988–998
Motea EA, Lee I, Berdis AJ (2012) Development of a ‘clickable’ non-natural nucleotide to visualize the replication of non-instructional DNA lesions. Nucleic Acids Res 40:2357–2367
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Choi, JS., Berdis, A. (2019). Artificial Nucleosides as Diagnostic Probes to Measure Translesion DNA Synthesis. In: Shank, N. (eds) Non-Natural Nucleic Acids. Methods in Molecular Biology, vol 1973. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9216-4_15
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DOI: https://doi.org/10.1007/978-1-4939-9216-4_15
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