RESUMEN

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent human carcinogen. Metabolic activation of NNK generates a number of DNA adducts including O(2)-methylthymidine (O(2)-Me-dT) and O(2)-[4-(3-pyridyl)-4-oxobut-1-yl]thymidine (O(2)-POB-dT). To investigate the biological effects of these O(2)-alkylthymidines in humans, we have replicated plasmids containing a site-specifically incorporated O(2)-Me-dT or O(2)-POB-dT in human embryonic kidney 293T (HEK293T) cells. The bulkier O(2)-POB-dT exhibited high genotoxicity and only 26% translesion synthesis (TLS) occurred, while O(2)-Me-dT was less genotoxic and allowed 55% TLS. However, O(2)-Me-dT was 20% more mutagenic (mutation frequency (MF) 64%) compared to O(2)-POB-dT (MF 53%) in HEK293T cells. The major type of mutations in each case was targeted T → A transversions (56% and 47%, respectively, for O(2)-Me-dT and O(2)-POB-dT). Both lesions induced a much lower frequency of T → G, the dominant mutation in bacteria. siRNA knockdown of the TLS polymerases (pols) indicated that pol η, pol ζ, and Rev1 are involved in the lesion bypass of O(2)-Me-dT and O(2)-POB-dT as the TLS efficiency decreased with knockdown of each pol. In contrast, MF of O(2)-Me-dT was decreased in pol ζ and Rev1 knockdown cells by 24% and 25%, respectively, while for O(2)-POB-dT, it was decreased by 44% in pol ζ knockdown cells, indicating that these TLS pols are critical for mutagenesis. Additional decrease in both TLS efficiency and MF was observed in cells deficient in pol ζ plus other Y-family pols. This study provided important mechanistic details on how these lesions are bypassed in human cells in both error-free and error-prone manner.

Asunto(s)

Carcinógenos/toxicidad , Daño del ADN , ADN Polimerasa Dirigida por ADN/fisiología , Mutágenos/toxicidad , Nicotiana/química , Nitrosaminas/toxicidad , Aductos de ADN/síntesis química , ADN Polimerasa Dirigida por ADN/genética , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Mutagénesis/efectos de los fármacos , Mutación , ARN Interferente Pequeño/genética

RESUMEN

The roles of translesion synthesis (TLS) DNA polymerases in bypassing the C8-2'-deoxyguanosine adduct (dG-C8-IQ) formed by 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), a highly mutagenic and carcinogenic heterocyclic amine found in cooked meats, were investigated. Three plasmid vectors containing the dG-C8-IQ adduct at the G1-, G2- or G3-positions of the NarI site (5'-G1G2CG3CC-3') were replicated in HEK293T cells. Fifty percent of the progeny from the G3 construct were mutants, largely G→T, compared to 18% and 24% from the G1 and G2 constructs, respectively. Mutation frequency (MF) of dG-C8-IQ was reduced by 38-67% upon siRNA knockdown of pol κ, whereas it was increased by 10-24% in pol η knockdown cells. When pol κ and pol ζ were simultaneously knocked down, MF of the G1 and G3 constructs was reduced from 18% and 50%, respectively, to <3%, whereas it was reduced from 24% to <1% in the G2 construct. In vitro TLS using yeast pol ζ showed that it can extend G3*:A pair more efficiently than G3*:C pair, but it is inefficient at nucleotide incorporation opposite dG-C8-IQ. We conclude that pol κ and pol ζ cooperatively carry out the majority of the error-prone TLS of dG-C8-IQ, whereas pol η is involved primarily in its error-free bypass.

Asunto(s)

Aductos de ADN , ADN Polimerasa Dirigida por ADN/fisiología , Desoxiguanosina/análogos & derivados , Mutágenos , Quinolinas , ADN/biosíntesis , Aductos de ADN/química , Desoxiguanosina/química , Células HEK293 , Humanos , Mutágenos/química , Tasa de Mutación , Quinolinas/química

RESUMEN

Abasic [apurinic/apyrimidinic (AP)] sites are the most common DNA damages, opposite which dAMP is frequently inserted ('A-rule') in Escherichia coli. Nucleotide insertion opposite the AP-site in eukaryotic cells depends on the assay system and the type of cells. Accordingly, a 'C-rule', 'A-rule', or the lack of specificity has been reported. DNA sequence context also modulates nucleotide insertion opposite AP-site. Herein, we have compared replication of tetrahydrofuran (Z), a stable analog of AP-site, in E. coli and human embryonic kidney 293T cells in two different sequences. The efficiency of translesion synthesis or viability of the AP-site construct in E. coli was less than 1%, but it was 7- to 8-fold higher in the GZGTC sequence than in the GTGZC sequence. The difference in viability increased even more in pol V-deficient strains. Targeted one-base deletions occurred in 63% frequency in the GZG and 68% frequency in GZC sequence, which dropped to 49% and 21%, respectively, upon induction of SOS. The full-length products with SOS primarily involved dAMP insertion opposite the AP-site, which occurred in 49% and 71% frequency, respectively, in the GZG and GZC sequence. dAMP insertion, largely carried out by pol V, was more efficient when the AP-site was a stronger replication block. In contrast to these results in E. coli, viability was 2 to 3 orders of magnitude higher in human cells, and the 'A-rule' was more rigidly followed. The AP-site in the GZG and GZC sequences gave 76% and 89%, respectively, Z → T substitutions. In human cells, targeted one-base deletion was undetectable, and dTMP>dCMP were the next preferred nucleotides inserted opposite Z. siRNA knockdown of Rev1 or pol ζ established that both these polymerases are vital for AP-site bypass, as demonstrated by 36-67% reduction in bypass efficiency. However, neither polymerase was indispensable, suggesting roles of additional DNA polymerases in AP-site bypass in human cells.

Asunto(s)

Daño del ADN , Escherichia coli/genética , Línea Celular , Replicación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Escherichia coli/metabolismo , Técnicas de Silenciamiento del Gen , Vectores Genéticos/genética , Células HEK293 , Humanos , Mutación , Nucleotidiltransferasas/metabolismo , Respuesta SOS en Genética

RESUMEN

3-Nitrobenzanthrone (3-NBA), a potent mutagen and suspected human carcinogen, is a common environmental pollutant. The genotoxicity of 3-NBA has been associated with its ability to form DNA adducts, including N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone (C8-dG-ABA). To investigate the molecular mechanism of C8-dG-ABA mutagenesis in human cells, we have replicated a plasmid containing a single C8-dG-ABA in human embryonic kidney 293T (HEK293T) cells, which yielded 14% mutant progeny. The major types of mutations induced by C8-dG-ABA were G→T>G→A>G→C. siRNA knockdown of the translesion synthesis (TLS) DNA polymerases (pols) in HEK293T cells indicated that pol η, pol κ, pol ι, pol ζ, and Rev1 each have a role in replication across this adduct. The extent of TLS was reduced with each pol knockdown, but the largest decrease (of ∼55% reduction) in the level of TLS occurred in cells with knockdown of pol ζ. Pol η and pol κ were considered the major contributors of the mutagenic TLS, because the mutation frequency (MF) decreased by 70%, when these pols were simultaneously knocked down. Rev1 also is important for mutagenesis, as reflected by the 60% reduction in MF upon Rev1 knockdown, but it probably plays a noncatalytic role by physically interacting with the other two Y-family pols. In contrast, pol ζ appeared to be involved in the error-free bypass of the lesion, because MF increased by 60% in pol ζ knockdown cells. These results provide important mechanistic insight into the bypass of the C8-dG-ABA adduct.

Asunto(s)

Benzo(a)Antracenos/toxicidad , ADN Polimerasa Dirigida por ADN/metabolismo , Desoxiguanosina/análogos & derivados , Proteínas Nucleares/metabolismo , Nucleotidiltransferasas/metabolismo , Carcinógenos Ambientales/toxicidad , ADN Polimerasa Dirigida por ADN/química , Desoxiguanosina/toxicidad , Regulación Enzimológica de la Expresión Génica/fisiología , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Estructura Molecular , Mutación , Proteínas Nucleares/genética , Nucleotidiltransferasas/genética , Interferencia de ARN , ARN Interferente Pequeño

RESUMEN

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.

Asunto(s)

Desoxiadenosinas/genética , Desoxiguanosina/análogos & derivados , Escherichia coli/genética , Mutágenos , ADN Polimerasa I/genética , ADN Polimerasa beta/genética , Desoxiguanosina/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Nucleótidos/metabolismo

RESUMEN

To investigate the biological effects of the O(2)-alkylthymidines induced by the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), we have replicated a plasmid containing O(2)-methylthymidine (O(2)-Me-dT) or O(2)-[4-(3-pyridyl-4-oxobut-1-yl]thymidine (O(2)-POB-dT) in Escherichia coli with specific DNA polymerase knockouts. High genotoxicity of the adducts was manifested in the low yield of transformants from the constructs, which was 2-5% in most strains but increased 2-4-fold with SOS. In the SOS-induced wild type E. coli, O(2)-Me-dT and O(2)-POB-dT induced 21% and 56% mutations, respectively. For O(2)-POB-dT, the major type of mutation was T → G followed by T → A, whereas for O(2)-Me-dT, T → G and T → A occurred in equal frequency. For both lesions, T → C also was detected in low frequency. The T → G mutation was reduced in strains with deficiency in any of the three SOS polymerases. By contrast, T → A was abolished in the pol V(-) strain, while its frequency in other strains remained unaltered. This suggests that pol V was responsible for the T → A mutations. The potent mutagenicity of these lesions may be related to NNK mutagenesis and carcinogenesis.

Asunto(s)

ADN Polimerasa Dirigida por ADN/genética , Escherichia coli/genética , Isoenzimas/genética , Nicotiana/química , Nitrosaminas/efectos adversos , Plásmidos/genética , Respuesta SOS en Genética/efectos de los fármacos , Carcinógenos/toxicidad , Cromatografía Líquida de Alta Presión , Recuento de Colonia Microbiana , Aductos de ADN/química , Aductos de ADN/genética , Aductos de ADN/metabolismo , Daño del ADN/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , ADN Polimerasa Dirigida por ADN/metabolismo , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Técnicas de Inactivación de Genes , Isoenzimas/metabolismo , Mutagénesis , Mutágenos/efectos adversos , Mutágenos/toxicidad , Nitrosaminas/toxicidad , Plásmidos/metabolismo , Piridinas/química , Piridinas/metabolismo , Espectrometría de Masa por Ionización de Electrospray , Timidina/análogos & derivados , Timidina/genética , Timidina/metabolismo

RESUMEN

8,5'-Cyclopurines, making up an important class of ionizing radiation-induced tandem DNA damage, are repaired only by nucleotide excision repair (NER). They accumulate in NER-impaired cells, as in Cockayne syndrome group B and certain Xeroderma Pigmentosum patients. A plasmid containing (5'S)-8,5'-cyclo-2'-deoxyguanosine (S-cdG) was replicated in Escherichia coli with specific DNA polymerase knockouts. Viability was <1% in the wild-type strain, which increased to 5.5% with SOS. Viability decreased further in a pol II(-) strain, whereas it increased considerably in a pol IV(-) strain. Remarkably, no progeny was recovered from a pol V(-) strain, indicating that pol V is absolutely required for bypassing S-cdG. Progeny analyses indicated that S-cdG is significantly mutagenic, inducing ~34% mutation with SOS. Most mutations were S-cdG → A mutations, though S-cdG → T mutation and deletion of 5'C also occurred. Incisions of purified UvrABC nuclease on S-cdG, S-cdA, and C8-dG-AP on a duplex 51-mer showed that the incision rates are C8-dG-AP > S-cdA > S-cdG. In summary, S-cdG is a major block to DNA replication, highly mutagenic, and repaired slowly in E. coli.

Asunto(s)

Reparación del ADN/genética , Replicación del ADN/efectos de los fármacos , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/genética , Desoxiguanosina/análogos & derivados , Escherichia coli/genética , Mutagénesis/genética , Reparación del ADN/efectos de los fármacos , Reparación del ADN/efectos de la radiación , Replicación del ADN/genética , Replicación del ADN/efectos de la radiación , ARN Polimerasas Dirigidas por ADN/antagonistas & inhibidores , Desoxiguanosina/química , Desoxiguanosina/genética , Desoxiguanosina/toxicidad , Escherichia coli/efectos de los fármacos , Escherichia coli/efectos de la radiación , Mutagénesis/efectos de los fármacos , Mutagénesis/efectos de la radiación , Respuesta SOS en Genética/efectos de los fármacos , Respuesta SOS en Genética/genética , Respuesta SOS en Genética/efectos de la radiación

RESUMEN

Mitomycin C induces both MC-mono-dG and cross-linked dG-adducts in vivo. Interstrand cross-linked (ICL) dG-MC-dG-DNA adducts can prevent strand separation. In Escherichia coli cells, UvrABC repairs ICL lesions that cause DNA bending. The mechanisms and consequences of NER of ICL dG-MC-dG lesions that do not induce DNA bending remain unclear. Using DNA fragments containing a MC-mono-dG or an ICL dG-MC-dG adduct, we found (i) UvrABC incises only at the strand containing MC-mono-dG adducts; (ii) UvrABC makes three types of incisions on an ICL dG-MC-dG adduct: type 1, a single 5' incision on 1 strand and a 3' incision on the other; type 2, dual incisions on 1 strand and a single incision on the other; and type 3, dual incisions on both strands; and (iii) the cutting kinetics of type 3 is significantly faster than type 1 and type 2, and all of 3 types of cutting result in producing DSB. We found that UvrA, UvrA+UvrB and UvrA+UvrB+UvrC bind to MC-modified DNA specifically, and we did not detect any UvrB- and UvrB+UvrC-DNA complexes. Our findings challenge the current UvrABC incision model. We propose that DSBs resulted from NER of ICL dG-MC-dG adducts contribute to MC antitumor activity and mutations.

Asunto(s)

Aductos de ADN/metabolismo , Reparación del ADN , Endodesoxirribonucleasas/metabolismo , Proteínas de Escherichia coli/metabolismo , Mitomicina/metabolismo , Modelos Genéticos , Aductos de ADN/química , Roturas del ADN de Doble Cadena , Mitomicina/química

RESUMEN

1-nitropyrene, the most abundant nitro polycyclic aromatic hydrocarbon in diesel emissions, has been found to react with DNA to form predominantly N-(deoxyguanosin-8-yl)-1-aminopyrene (dGAP). This bulky adduct has been shown to induce genetic mutations, which may implicate Y-family DNA polymerases in its bypass in vivo. To establish a kinetic mechanism for the bypass of such a prototype single-base lesion, we employed pre-steady-state kinetic methods to investigate individual nucleotide incorporations upstream, opposite, and downstream from a site-specifically placed dGAP lesion catalyzed by Sulfolobus solfataricus DNA polymerase IV (Dpo4), a model Y-family DNA polymerase. Dpo4 was able to bypass dGAP but paused strongly at two sites: opposite the lesion and immediately downstream from the lesion. Both nucleotide incorporation efficiency and fidelity decreased significantly at the pause sites, especially during extension of the bypass product. Interestingly, a 4-fold tighter binding affinity of damaged DNA to Dpo4 promoted catalysis through putative interactions between the active site residues of Dpo4 and 1-aminopyrene moiety at the first pause site. In the presence of a DNA trap, the kinetics of nucleotide incorporation at these sites was biphasic in which a small, fast phase preceded a larger, slow phase. In contrast, only a large, fast phase was observed during nucleotide incorporation at non-pause sites. Our kinetic studies support a general kinetic mechanism for lesion bypass catalyzed by numerous DNA polymerases.

Asunto(s)

Proteínas Arqueales/química , Aductos de ADN/química , ADN de Archaea/química , ADN Polimerasa Dirigida por ADN/química , Pirenos/química , Sulfolobus solfataricus/enzimología , Proteínas Arqueales/genética , Catálisis , Aductos de ADN/genética , Aductos de ADN/metabolismo , Daño del ADN/fisiología , ADN de Archaea/genética , ADN de Archaea/metabolismo , ADN Polimerasa Dirigida por ADN/genética , Cinética , Mutación , Sulfolobus solfataricus/genética , Emisiones de Vehículos

RESUMEN

Thymine glycol (Tg), 5,6-dihydroxy-5,6-dihydrothymine, is formed in DNA by the reaction of thymine with reactive oxygen species. The 5R Tg lesion was incorporated site-specifically into 5'-d(G(1)T(2)G(3)C(4)G(5)Tg(6)G(7)T(8)T(9)T(10)G(11)T(12))-3'; Tg = 5R Tg. The Tg-modified oligodeoxynucleotide was annealed with either 5'-d(A(13)C(14)A(15)A(16)A(17)C(18)A(19)C(20)G(21)C(22)A(23)C(24))-3', forming the Tg(6) x A(19) base pair, corresponding to the oxidative damage of thymine in DNA, or 5'-d(A(13)C(14)A(15)A(16)A(17)C(18)G(19)C(20)G(21)C(22)A(23)C(24))-3', forming the mismatched Tg(6) x G(19) base pair, corresponding to the formation of Tg following oxidative damage and deamination of 5-methylcytosine in DNA. At 30 degrees C, the equilibrium ratio of cis-5R,6S:trans-5R,6R epimers was 7:3 for the duplex containing the Tg(6) x A (19) base pair. In contrast, for the duplex containing the Tg(6) x G(19) base pair, the cis-5R,6S:trans-5R,6R equilibrium favored the cis-5R,6S epimer; the level of the trans-5R,6R epimer remained below the level of detection by NMR. The data suggested that Tg disrupted hydrogen bonding interactions, either when placed opposite to A(19) or G(19). Thermodynamic measurements indicated a 13 degrees C reduction of T(m) regardless of whether Tg was placed opposite dG or dA in the complementary strand. Although both pairings increased the free energy of melting by 3 kcal/mol, the melting of the Tg x G pair was more enthalpically favored than was the melting of the Tg x A pair. The observation that the position of the equilibrium between the cis-5R,6S and trans-5R,6R thymine glycol epimers in duplex DNA was affected by the identity of the complementary base extends upon observations that this equilibrium modulates the base excision repair of Tg [Ocampo-Hafalla, M. T.; Altamirano, A.; Basu, A. K.; Chan, M. K.; Ocampo, J. E.; Cummings, A., Jr.; Boorstein, R. J.; Cunningham, R. P.; Teebor, G. W. DNA Repair (Amst) 2006, 5, 444-454].

Asunto(s)

ADN/química , Timina/análogos & derivados , Conformación de Carbohidratos , ADN/metabolismo , Desoxirribosa/química , Desoxirribosa/metabolismo , Resonancia Magnética Nuclear Biomolecular/métodos , Conformación de Ácido Nucleico , Oligonucleótidos/química , Termodinámica , Timina/química , Timina/metabolismo