Mutagenesis of 8-oxoguanine adjacent to an abasic site in simian kidney cells: tandem mutations and enhancement of G-->T transversions.
Clustered DNA damages are well-established characteristics of ionizing radiation. As a model clustered lesion in the same strand of DNA, we have evaluated the mutagenic potential of 8-oxoguanine (8-oxoG) adjacent to a uracil in simian kidney cells using a phagemid vector. The uracil residue would be excised by the enzyme uracil DNA glycosylase in vivo generating an abasic site (AP site). A solitary uracil in either GUGTC or GTGUC sequence context provided >60% progeny containing GTGTC indicating that dAMP incorporation opposite the AP site or uracil occurred, but a >30% population showed replacement of U by A, C, or G, which suggests that dTMP, dGMP, or dCMP incorporation also occurred, respectively, opposite the AP site. While the preference for targeted base substitutions at the GUG site was T >> C > A > G, the same at the GUC site was T >> A > C > G. We conclude that base incorporation opposite an AP site is sequence-dependent. For 8-oxoG, as compared to 23-24% G-->T mutants from a single 8-oxoG in a TG(8-oxo)T sequence context, the tandem lesions UG(8-oxo)T and TG(8-oxo)U generated approximately 60 and >85% progeny, respectively, that did not contain the TGT sequence. A significant fraction of tandem mutations were detected when uracil was adjacent to 8-oxoG. What we found most interesting is that the total targeted G(8-oxo)-->T transversions that included both single and tandem mutations at the TG(8-oxo)U site was nearly 60% relative to about 30% at the UG(8-oxo)T site. A higher mutational frequency at the TG(8-oxo)U sequence may arise from a change in DNA polymerase that is more error prone. Thermal melting experiments showed that the Tm for the 8-oxoG:C pair in the TG(8-oxo)(AP*) sequence in a 12-mer was lower than the same in a (AP*)G(8-oxo)T 12-mer with deltadeltaG 0.8 kcal/mol (where AP* represents tetrahydrofuran, the model abasic site). When the 8-oxoG:C pair in each sequence was compared with a 8-oxoG:A pair, the former was found to be more stable than the latter. The preference for C over A opposite 8-oxoG for the (AP*)G(8-oxo)T 12-mer duplex with a deltadeltaG of 1.6 kcal/mol dropped to 0.4 kcal/mol in the TG(8-oxo)(AP*) 12-mer duplex. This suggests that the polymerase discrimination to incorporate dCMP over dAMP would be less efficient in the TG(8-oxo)(AP*) sequence relative to (AP*)G(8-oxo)T. Additionally, the efficiency of recognition and excision of A opposite 8-oxoG by a mismatch repair protein may be impaired in the TG(8-oxo)(AP*) sequence context.
Kalam
M Abul
MA
Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269, USA.
Basu
Ashis K
AK
D000818:Animals; D019556:COS Cells; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D004247:DNA / Q000235:genetics; D005720:Gamma Rays; D005822:Genetic Vectors; D006147:Guanine / Q000031:analogs & derivatives* / Q000528:radiation effects / Q000633:toxicity; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D016296:Mutagenesis; D009153:Mutagens / Q000528:radiation effects / Q000633:toxicity*; D015345:Oligonucleotide Probes / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature; D013816:Thermodynamics; D014498:Uracil / Q000378:metabolism
D009153:Mutagens; D015345:Oligonucleotide Probes; C024829:8-hydroxyguanine; D014498:Uracil; D006147:Guanine; D004247:DNA