Ionizing radiation and aerobic metabolism generate many lesions in DNA including 8,5'-cyclopurine-2'-deoxynucleotides. These complex lesions, including (5'S)-8,5'-cyclo-2'-deoxyadenosine (S-cdA) and (5'S)-8,5'-cyclo-2'-deoxyguanosine (S-cdG), have been shown to be genotoxic and mutagenic in Escherichia coli and human cells. In both E. coli and human cells, S-cdG is more mutagenic than S-cdA. To better understand replication past these damages, we measured the rate of insertion of dNTPs opposite S-cdG and S-cdA as well as further extension by E. coli DNA polymerase I (KF (exo-)), Sulfolobus solfataricus DNA polymerase IV (Dpo4) and E. coli DNA polymerase IV (pol IV). KF (exo-) stalled at the lesion site and was unable to form a full-length extension product even when high concentration of the enzyme was used for a prolonged time period. The steady state kinetic parameters showed error-free replication of both S-cdA and S-cdG by KF (exo-). In contrast, although S-cdA and S-cdG were major blocks of DNA replication by Dpo4 and pol IV, these Y-family DNA polymerases were able to extend a small fraction of the primer to a full-length product. Steady-state kinetic parameters of dNTP insertion by Dpo4 showed that the correct nucleotide dTMP is preferentially incorporated opposite S-cdA, whereas for S-cdG the wrong nucleotide dTMP is incorporated preferentially over dCMP. The results of this study are consistent with the strong replication blocking nature of S-cdA and S-cdG, and their ability to initiate error-prone synthesis by Y-family DNA polymerases.[Supported by NIEHS grant ES09127]

In vitro translesion synthesis of 8,5'-cyclopurine-2'-deoxynucleosides by exo-free Klenow fragment, DNA polymerase IV, and Dpo4

247th National Meeting (2014)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.