title: Mutagenicity and genotoxicity of (5'S)-8,5'-cyclo-2'-deoxyadenosine in Escherichia coli and replication of (5'S)-8,5'-cyclopurine-2'-deoxynucleosides in vitro by DNA polymerase IV, exo-free Klenow fragment, and Dpo4.
abstract: Reactive oxygen species generate many lesions in DNA, including R and S diastereomers of 8,5'-cyclo-2'-deoxyadenosine (cdA) and 8,5'-cyclo-2'-deoxyguanosine (cdG). Herein, the result of replication of a plasmid containing S-cdA in Escherichia coli is reported. S-cdA was found mutagenic and highly genotoxic. Viability and mutagenicity of the S-cdA construct were dependent on functional pol V, but mutational frequencies (MFs) and types varied in pol II- and pol IV-deficient strains relative to the wild-type strain. Both S-cdA → T and S-cdA → G substitutions occurred in equal frequency in wild-type E. coli, but the frequency of S-cdA → G dropped in pol IV-deficient strain, especially when being SOS induced. This suggests that pol IV plays a role in S-cdA → G mutations. MF increased significantly in pol II-deficient strain, suggesting pol II's likely role in error-free translesion synthesis. Primer extension and steady-state kinetic studies using pol IV, exo-free Klenow fragment (KF (exo(-))), and Dpo4 were performed to further assess the replication efficiency and fidelity of S-cdA and S-cdG. Primer extension by pol IV mostly stopped before the lesion, although a small fraction was extended opposite the lesion. Kinetic studies showed that pol IV incorporated dCMP almost as efficiently as dTMP opposite S-cdA, whereas it incorporated the correct nucleotide dCMP opposite S-cdG 10-fold more efficiently than any other dNMP. Further extension of each lesion containing pair, however, was very inefficient. These results are consistent with the role of pol IV in S-cdA → G mutations in E. coli. KF (exo(-)) was also strongly blocked by both lesions, but it could slowly incorporate the correct nucleotide opposite them. In contrast, Dpo4 could extend a small fraction of the primer to a full-length product on both S-cdG and S-cdA templates. Dpo4 incorporated dTMP preferentially opposite S-cdA over the other dNMPs, but the discrimination was only 2- to 8-fold more proficient. Further extension of the S-cdA:T and S-cdA:C pair was not much different. For S-cdG, conversely, the wrong nucleotide, dTMP, was incorporated more efficiently than dCMP, although one-base extension of the S-cdG:T pair was less efficient than the S-cdG:C pair. S-cdG, therefore, has the propensity to cause G → A transition, as was reported to occur in E. coli. 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.
authors.affiliation: Department of Chemistry, University of Connecticut , Storrs, Connecticut 06269, United States.
authors.forename: Varsha
authors.initials: V
authors.lastname: Pednekar
authors.forename: Savithri
authors.initials: S
authors.lastname: Weerasooriya
authors.forename: Vijay P
authors.initials: VP
authors.lastname: Jasti
authors.forename: Ashis K
authors.initials: AK
authors.lastname: Basu
mesh_terms: D004256:DNA Polymerase I / Q000235:genetics; D019951:DNA Polymerase beta / Q000235:genetics; D003839:Deoxyadenosines / Q000235:genetics*; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics; D009153:Mutagens; D009711:Nucleotides / Q000378:metabolism
chemical_list: D003839:Deoxyadenosines; D029968:Escherichia coli Proteins; D009153:Mutagens; D009711:Nucleotides; C050888:8,5'-cyclo-2'-deoxyguanosine; C056752:8,5'-cyclo-2'-deoxyadenosine; C509838:Dpo4 protein, E coli; D004256:DNA Polymerase I; D019951:DNA Polymerase beta; D003849:Deoxyguanosine