title: In vitro replication and thermodynamic studies of methylation and oxidation modifications of 6-thioguanine.
abstract: The cytotoxic effects of thiopurine drugs are mostly exerted through the formation of thioguanine nucleotide and its subsequent incorporation into DNA. The 6-thioguanine (6-TG) in DNA can be converted to S6-methylthio-2-aminopurine (2-AP-6-SCH3) and 2-aminopurine-6-sulfonic acid (2-AP-6-SO3H) upon reaction with S-adenosyl-L-methionine and irradiation with UVA light, respectively. Here we prepared oligodeoxynucleotides (ODNs) harboring a 6-TG, 2-AP-6-SCH3 or 2-AP-6-SO3H at a defined site and examined, by using LC-MS/MS, the in vitro replication of these substrates with yeast polymerase eta and Klenow fragment (KF-). Our results revealed that 2-AP-6-SCH3 could be bypassed by KF-, with significant misincorporation of thymine opposite the lesion. The 2-AP-6-SO3H, however, blocked markedly the nucleotide insertion by KF-. Yeast pol eta could bypass all three modified nucleosides; although dCMP was inserted preferentially, we found substantial misincorporation of dTMP and dAMP opposite 2-AP-6-SCH3 and 2-AP-6-SO3H, respectively. Moreover, both KF- and yeast pol eta induced a considerable amount of -2 frameshift products from the replication of 2-AP-6-SCH3- and 2-AP-6-SO3H-bearing substrates. Our results also underscored the importance of measuring the relative ionization efficiencies of replication products in the accurate quantification of these products by LC-MS/MS. Moreover, thermodynamic studies revealed that 2-AP-6-SCH3 and 2-AP-6-SO3H could cause more destabilization to duplex DNA than 6-TG. Taken together, the results from this study shed important new light on the biological implications of the two metabolites of 6-TG.
authors.affiliation: Environmental Toxicology Graduate Program, University of California, Riverside, CA 92521-0403, USA.
authors.forename: Chunang
authors.initials: C
authors.lastname: Gu
authors.forename: Yinsheng
authors.initials: Y
authors.lastname: Wang
mesh_terms: D015075:2-Aminopurine / Q000031:analogs & derivatives / Q000737:chemistry; D002853:Chromatography, Liquid; D019175:DNA Methylation; D004256:DNA Polymerase I / Q000378:metabolism; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D015122:Mercaptopurine / Q000031:analogs & derivatives / Q000737:chemistry; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry; D010084:Oxidation-Reduction; D053719:Tandem Mass Spectrometry; D013816:Thermodynamics; D013866:Thioguanine / Q000737:chemistry* / Q000378:metabolism; D000099270:Y-Family DNA Polymerases
chemical_list: D015075:2-Aminopurine; D004256:DNA Polymerase I; D004259:DNA-Directed DNA Polymerase; D015122:Mercaptopurine; D009838:Oligodeoxyribonucleotides; D013866:Thioguanine; C521424:2-aminopurine-6-sulfonic acid; C521423:S6-methylthio-2-aminopurne; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases