A new approach utilizing real-time qPCR to detect in vitro base excision repair

DNA Repair
Honghai ZhangDexi Chen

Abstract

DNA lesions in mammalian cells may be induced by reactive oxygen species, alkylation, and ionizing radiation. This damage can then be repaired via the base excision repair (BER) pathway, which includes single strand break repair (SSBR). Thus, the BER (SSBR) pathway plays a critical role in maintaining genomic integrity, and may help us to better understand the mechanisms of aging, tumor formation, and degenerative diseases. AP site (apurinic/apyrimidinic site) or damaged base excision, nucleotide insertion and ligation are included in the BER (SSBR) pathway, which are facilitated by different enzymes at each step. Most previous in vitro BER studies have used modified radiolabeled (32)P oligonucleotide substrates. Which is a very conventional method for in vitro BER assay. However, the use of radioactive isotope material was limited in various laboratories which are unable to handle radioactive hazard. In this study, we developed a novel technique using real-time quantitative PCR (qPCR) to quantify BER activity in in vitro assays. Single strand breaks, DNA ligase activity, and glycosylase activity were detected to establish the feasibility and advantages of this qPCR technique for in vitro BER profiling.

References

Jun 1, 1995·Free Radical Biology & Medicine·A P Breen, J A Murphy
Apr 19, 1996·The Journal of Biological Chemistry·G FrosinaE Dogliotti
Aug 8, 1997·The Journal of Biological Chemistry·K B Beckman, B N Ames
Mar 31, 1999·Free Radical Research·C M GedikA R Collins
Sep 16, 1999·Mutation Research·D L CroteauV A Bohr
Jan 19, 2000·Proceedings of the National Academy of Sciences of the United States of America·H AtamnaB N Ames
Dec 18, 2001·Environmental and Molecular Mutagenesis·S MitraT K Hazra
Mar 23, 2002·Molecular and Cellular Biology·Richard M TaylorKeith W Caldecott
Apr 12, 2002·Gene·Vilhelm A BohrNadja C de Souza-Pinto
Apr 13, 2002·Photochemistry and Photobiology·David L MitchellJean Cadet
Nov 13, 2002·Mutation Research·Bhaskar S MandavilliBennett Van Houten
Oct 3, 2003·The Journal of Biological Chemistry·Hong DouTapas K Hazra
Nov 26, 2003·Mutation Research·Geir SlupphaugHans E Krokan
Jul 29, 2004·DNA Repair·Ari Barzilai, Ken-Ichi Yamamoto
Jul 8, 2005·Journal of Cerebral Blood Flow and Metabolism : Official Journal of the International Society of Cerebral Blood Flow and Metabolism·Wenjin LiJun Chen
Sep 15, 2005·Cancer Letters·Christine L PowellIvan Rusyn
Nov 25, 2006·DNA Repair·Grigory L Dianov, Jason L Parsons
Feb 19, 2008·Journal of Virological Methods·Tibor TakácsAttila Sebe
Aug 30, 2008·Journal of Virological Methods·Mohamed AitichouSofi Ibrahim
Aug 25, 2009·Toxicology and Applied Pharmacology·S M WnekA J Gandolfi

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Citations

Nov 3, 2012·The Journal of Biological Chemistry·Anna-Kate FowlerInna I Kruman
Jul 3, 2016·Journal of Pharmaceutical and Biomedical Analysis·Justyna WiczkJanusz Rak
Apr 19, 2018·Human Molecular Genetics·Ling-Yueh HuChen-Yang Shen

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