Activity of DNA polymerase κ across the genome in human fibroblasts.

DNA polymerase κ (Polκ) is a specialized polymerase that has multiple cellular roles such as translesion DNA synthesis, replication of repetitive sequences, and nucleotide excision repair. We have developed a method for capturing DNA synthesized by Polκ utilizing a Polκ-specific substrate, N2-(4-ethynylbenzyl)-2'-deoxyguanosine (EBndG). After shearing of the DNA into 200 to 500 bp lengths, the EBndG-containing DNA was covalently bound to biotin using the Cu(I)-catalyzed alkyne-azide cycloaddition reaction and isolated with streptavidin beads. Isolated DNA was then ligated to adaptors, followed by PCR amplification and next-generation sequencing to generate genome-wide repair maps. We have termed this method polymerase κ sequencing. Here, we present the human genome maps for Polκ activity in an undamaged cell line. We found that Polκ activity was enhanced in GC-rich regions, euchromatin regions, the promoter of genes, and in DNA that is replicated early in the S phase.

Active Site Interactions Impact Phosphoryl Transfer during Replication of Damaged and Undamaged DNA by Escherichia coli DNA Polymerase I.

Replicative DNA polymerases are able to discriminate between very similar substrates with high accuracy. One mechanism by which E. coli DNA polymerase I checks for Watson-Crick geometry is through a hydrogen bonding fork between Arg668 and the incoming dNTP and the minor groove of the primer terminus. The importance of the Arg-fork was examined by disrupting it with either a guanine to 3-deazaguanine substitution at the primer terminus or the use of a carbocyclic deoxyribose analog of dUTP. Using thio-substituted dNTPs and differential quench techniques, we determined that when the Arg-fork was disrupted, the rate-limiting step changed from a conformational change to phosphodiester bond formation. This result indicates that Arg668 is involved in the phosphoryl transfer step. We examined the role of the Arg-fork in the replication of four DNA damaged templates, O6-methylguanine (O6-mG), 8-oxo-7,8-dihydroguanine (oxoG), O2-[4-(3-pyridyl)-4-oxobutyl]thymine (O2-POB-T), and N2-[(7S,8R,9S,10R)-7,8,9,10-tetrahydro-8,9,10-trihydroxybenzo[a]pyren-7-yl]-guanine (N2-BP-G). In general, the guanine to 3-deazaguanine substitution caused a decrease in kpol that was proportional to kpol over five orders of magnitude. The linear relationship indicates that the Arg668-fork helps catalyze phosphoryl transfer by the same mechanism with all the substrates. Exceptions to the linear relationship were the incorporations of dTTP opposite G, oxoG, and O6mG, which showed large decreases in kpol, similar to that exhibited by the Watson-Crick base pairs. It was proposed that the incorporation of dTTP opposite G, oxoG, and O6mG occurred via Watson-Crick-like structures.

Honokiol Inhibits DNA Polymerases ß and λ and Increases Bleomycin Sensitivity of Human Cancer Cells.

A major concept to sensitize cancer cells to DNA damaging agents is by inhibiting proteins in the DNA repair pathways. X-family DNA polymerases play critical roles in both base excision repair (BER) and nonhomologous end joining (NHEJ). In this study, we examined the effectiveness of honokiol to inhibit human DNA polymerase ß (pol ß), which is involved in BER, and DNA polymerase λ (pol λ), which is involved in NHEJ. Kinetic analysis with purified polymerases showed that honokiol inhibited DNA polymerase activity. The inhibition mode for the polymerases was a mixed-function noncompetitive inhibition with respect to the substrate, dCTP. The X-family polymerases, pol ß and pol λ, were slightly more sensitive to inhibition by honokiol based on the Ki value of 4.0 µM for pol ß, and 8.3 µM for pol λ, while the Ki values for pol η and Kf were 20 and 26 µM, respectively. Next we extended our studies to determine the effect of honokiol on the cytotoxicity of bleomycin and temozolomide in human cancer cell lines A549, MCF7, PANC-1, UACC903, and normal blood lymphocytes (GM12878). Bleomycin causes both single strand DNA damage that is repaired by BER and double strand breaks that are repaired by NHEJ, while temozolomide causes methylation damage repaired by BER and O6-alkylguanine-DNA alkyltransferase. The greatest effects were found with the honokiol and bleomycin combination in MCF7, PANC-1, and UACC903 cells, in which the EC50 values were decreased 10-fold. The temozolomide and honokiol combination was less effective; the EC50 values decreased three-fold due to the combination. It is hypothesized that the greater effect of honokiol on bleomycin is due to inhibition of the repair of the single strand and double strand damage. The synergistic activity shown by the combination of bleomycin and honokiol suggests that they can be used as combination therapy for treatment of cancer, which will decrease the therapeutic dosage and side effects of bleomycin.

Mutagenic Replication of N2-Deoxyguanosine Benzo[a]pyrene Adducts by Escherichia coli DNA Polymerase I and Sulfolobus solfataricus DNA Polymerase IV.

Benzo[a]pyrene, a potent human carcinogen, is metabolized in vivo to a diol epoxide that reacts with the N2-position of guanine to produce N2-BP-dG adducts. These adducts are mutagenic causing G to T transversions. These adducts block replicative polymerases but can be bypassed by the Y-family translesion synthesis polymerases. The mechanisms by which mutagenic bypass occurs is not well-known. We have evaluated base pairing structures using atomic substitution of the dNTP with two stereoisomers, 2'-deoxy-N-[(7R,8S,9R,10S)-7,8,9,10-tetrahydro-7,8,9-trihydroxybenzo[a]pyren-10-yl]guanosine and 2'-deoxy-N-[(7S,8R,9S,10R)-7,8,9,10-tetrahydro-7,8,9-trihydroxybenzo[a]pyren-10-yl]guanosine. We have examined the kinetics of incorporation of 1-deaza-dATP, 7-deaza-dATP, 2'-deoxyinosine triphosphate, and 7-deaza-dGTP, analogues of dATP and dGTP in which single atoms are changed. Changes in rate will occur if that atom provided a critical interaction in the transition state of the reaction. We examined two polymerases, Escherichia coli DNA polymerase I (Kf) and Sulfolobus solfataricus DNA polymerase IV (Dpo4), as models of a high fidelity and TLS polymerase, respectively. We found that with Kf, substitution of the nitrogens on the Watson-Crick face of the dNTPs resulted in decreased rate of reactions. This result is consistent with a Hoogsteen base pair in which the template N2-BP-dG flipped from the anti to syn conformation. With Dpo4, while the substitution did not affect the rate of reaction, the amplitude of the reaction decreased with all substitutions. This result suggests that Dpo4 bypasses N2-BP-dG via Hoogsteen base pairs but that the flipped nucleotide can be either the dNTP or the template.

N2 -Substituted 2'-Deoxyguanosine Triphosphate Derivatives as Selective Substrates for Human DNA Polymerase κ.

N2 -Alkyl-2'-deoxyguanosine triphosphate (N2 -alkyl-dGTP) derivatives with methyl, butyl, benzyl, or 4-ethynylbenzyl substituents were prepared and tested as substrates for human DNA polymerases. N2 -Benzyl-dGTP was equal to dGTP as a substrate for DNA polymerase κ (pol κ), but was a poor substrate for pols ß, δ, η, ι, or ν. In vivo reactivity was evaluated through incubation of N2 -4-ethynylbenzyl-dG with wild-type and pol κ deficient mouse embryonic fibroblasts. CuAAC reaction with 5(6)-FAM-azide demonstrated that only cells containing pol κ were able to incorporate N2 -4-ethynylbenzyl-dG into the nucleus. This is the first instance of a Y-family-polymerase-specific dNTP, and this method could be used to probe the activity of pol κ in vivo.

Human DNA Polymerase ν Catalyzes Correct and Incorrect DNA Synthesis with High Catalytic Efficiency.

DNA polymerase ν (pol ν) is a low fidelity A-family polymerase with a putative role in interstrand cross-link repair and homologous recombination. We carried out pre-steady-state kinetic analysis to elucidate the kinetic mechanism of this enzyme. We found that the mechanism consists of seven steps, similar that of other A-family polymerases. pol ν binds to DNA with a Kd for DNA of 9.2 nm, with an off-rate constant of 0.013 s(-1)and an on-rate constant of 14 µm(-1) s(-1). dNTP binding is rapid with Kd values of 20 and 476 µm for the correct and incorrect dNTP, respectively. Pyrophosphorylation occurs with a Kd value for PPi of 3.7 mm and a maximal rate constant of 11 s(-1). Pre-steady-state kinetics, examination of the elemental effect using dNTPαS, and pulse-chase experiments indicate that a rapid phosphodiester bond formation step is flanked by slow conformational changes for both correct and incorrect base pair formation. These experiments in combination with computer simulations indicate that the first conformational change occurs with rate constants of 75 and 20 s(-1); rapid phosphodiester bond formation occurs with a Keq of 2.2 and 1.7, and the second conformational change occurs with rate constants of 2.1 and 0.5 s(-1), for correct and incorrect base pair formation, respectively. The presence of a mispair does not induce the polymerase to adopt a low catalytic conformation. pol ν catalyzes both correct and mispair formation with high catalytic efficiency.

DNA Polymerase ν Rapidly Bypasses O6-Methyl-dG but Not O6-[4-(3-Pyridyl)-4-oxobutyl-dG and O2-Alkyl-dTs.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent tobacco carcinogen that forms mutagenic DNA adducts including O6-methyl-2'-deoxyguanosine (O6-Me-dG), O6-[4-(3-pyridyl)-4-oxobut-1-yl]-dG (O6-POB-dG), O2-methylthymidine (O2-Me-dT), and O2-POB-dT. We evaluated the ability of human DNA polymerase ν to bypass this damage to evaluate the structural constraints on substrates for pol ν and to evaluate if there is kinetic evidence suggesting the in vivo activity of pol ν on tobacco-induced DNA damage. Presteady-state kinetic analysis has indicated that O6-Me-dG is a good substrate for pol ν, while O6-POB-dG and the O2-alkyl-dT adducts are poor substrates for pol ν. The reactivity with O6-Me-dG is high with a preference for dCTP > dGTP > dATP > dTTP. The catalytic activity of pol ν toward O6-Me-dG is high and can potentially be involved in its bypass in vivo. In contrast, pol ν is unlikely to bypass O6-POB-dG or the O2-alkyl-dTs in vivo.

DNA Polymerases η and ζ Combine to Bypass O(2)-[4-(3-Pyridyl)-4-oxobutyl]thymine, a DNA Adduct Formed from Tobacco Carcinogens.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) are important human carcinogens in tobacco products. They are metabolized to produce a variety 4-(3-pyridyl)-4-oxobutyl (POB) DNA adducts including O(2)-[4-(3-pyridyl)-4-oxobut-1-yl]thymidine (O(2)-POB-dT), the most abundant POB adduct in NNK- and NNN-treated rodents. To evaluate the mutagenic properties of O(2)-POB-dT, we measured the rate of insertion of dNTPs opposite and extension past O(2)-POB-dT and O(2)-Me-dT by purified human DNA polymerases η, κ, ι, and yeast polymerase ζ in vitro. Under conditions of polymerase in excess, polymerase η was most effective at the insertion of dNTPs opposite O(2)-alkyl-dTs. The time courses were biphasic suggesting the formation of inactive DNA-polymerase complexes. The kpol parameter was reduced approximately 100-fold in the presence of the adduct for pol η, κ, and ι. Pol η was the most reactive polymerase for the adducts due to a higher burst amplitude. For all three polymerases, the nucleotide preference was dATP > dTTP ≫ dGTP and dCTP. Yeast pol ζ was most effective in bypassing the adducts; the kcat/Km values were reduced only 3-fold in the presence of the adducts. The identity of the nucleotide opposite the O(2)-alkyl-dT did not significantly affect the ability of pol ζ to bypass the adducts. The data support a model in which pol η inserts ATP or dTTP opposite O(2)-POB-dT, and then, pol ζ extends past the adduct.

An unprecedented nucleic acid capture mechanism for excision of DNA damage.

DNA glycosylases that remove alkylated and deaminated purine nucleobases are essential DNA repair enzymes that protect the genome, and at the same time confound cancer alkylation therapy, by excising cytotoxic N3-methyladenine bases formed by DNA-targeting anticancer compounds. The basis for glycosylase specificity towards N3- and N7-alkylpurines is believed to result from intrinsic instability of the modified bases and not from direct enzyme functional group chemistry. Here we present crystal structures of the recently discovered Bacillus cereus AlkD glycosylase in complex with DNAs containing alkylated, mismatched and abasic nucleotides. Unlike other glycosylases, AlkD captures the extrahelical lesion in a solvent-exposed orientation, providing an illustration for how hydrolysis of N3- and N7-alkylated bases may be facilitated by increased lifetime out of the DNA helix. The structures and supporting biochemical analysis of base flipping and catalysis reveal how the HEAT repeats of AlkD distort the DNA backbone to detect non-Watson-Crick base pairs without duplex intercalation.

In vitro epsilon RNA-dependent protein priming activity of human hepatitis B virus polymerase.

Hepatitis B virus (HBV) replicates its DNA genome through reverse transcription of a pregenomic RNA (pgRNA) by using a multifunctional polymerase (HP). A critical function of HP is its specific recognition of a viral RNA signal termed ε (Hε) located on pgRNA, which is required for specific packaging of pgRNA into viral nucleocapsids and initiation of viral reverse transcription. HP initiates reverse transcription by using itself as a protein primer (protein priming) and Hε as the obligatory template. We have purified HP from human cells that retained Hε binding activity in vitro. Furthermore, HP purified as a complex with Hε, but not HP alone, displayed in vitro protein priming activity. While the HP-Hε interaction in vitro and in vivo required the Hε internal bulge, but not its apical loop, and was not significantly affected by the cap-Hε distance, protein priming required both the Hε apical loop and internal bulge, as well as a short distance between the cap and Hε, mirroring the requirements for RNA packaging. These studies have thus established new HBV protein priming and RNA binding assays that should greatly facilitate the dissection of the requirements and molecular mechanisms of HP-Hε interactions, RNA packaging, and protein priming.

Low fidelity bypass of O(2)-(3-pyridyl)-4-oxobutylthymine, the most persistent bulky adduct produced by the tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone by model DNA polymerases.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one of the most important human carcinogens. It is metabolized to produce a variety of methyl and 4-(3-pyridyl)-4-oxo-butyl (POB) DNA adducts. A potentially important POB adduct is O(2)-[4-(3-pyridyl)-4-oxobut-1-yl]thymidine (O(2)-POB-dT) because it is the most abundant POB adduct in NNK-treated rodents. To evaluate the mutagenic properties of O(2)-POB-dT, we measured the rate of insertion of dNTPs opposite and extension past both O(2)-POB-dT and O(2)-methylthymidine (O(2)-Me-dT) by two model polymerases, E. coli DNA polymerase I (Klenow fragment) with the proofreading exonuclease activity inactivated (Kf) and Sulfolobus solfataricus DNA polymerase IV (Dpo4). We found that the size of the alkyl chain only marginally affected the reactivity and that the specificity of adduct bypass was very low. The k(cat)/K(m) for the Kf catalyzed incorporation opposite and extension past the adducts was reduced ∼10(6)-fold when compared to undamaged DNA. Dpo4 catalyzed the incorporation opposite and extension past the adducts approximately 10(3)-fold more slowly than undamaged DNA. The dNTP specificity was less for Dpo4 than for Kf. In general, dA was the preferred base pair partner for O(2)-Me-dT and dT the preferred base pair partner for O(2)-POB-dT. With enzyme in excess over DNA, the time courses of the reactions showed a biphasic kinetics that indicates the formation inactive binary and ternary complexes.

Tobacco-specific nitrosamine-derived O2-alkylthymidines are potent mutagenic lesions in SOS-induced Escherichia coli.

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.

Characterization of dibenzo[a,l]pyrene-trans-11,12-diol (dibenzo[def,p]chrysene) glucuronidation by UDP-glucuronosyltransferases.

Dibenzo[a,l]pyrene (DB[a,l]P) (dibenzo[def,p]chrysene) is a highly carcinogenic polycyclic aromatic hydrocarbon (PAH) that has been identified in tobacco smoke and is found in our environment due to incomplete combustion of organic matter. Its metabolites are known to form stable DNA adducts in bacteria and mammalian cells, and can lead to tumors in animal models. Glucuronidation of major metabolites of DB[a,l]P by the uridine-5'-diphosphate glucuronosyltransferase (UGT) family of enzymes is an important route of detoxification of this pro-carcinogen. The focus of the current study was to characterize the glucuronidation of the pro-carcinogenic enantiomers DB[a,l]P-(+)-trans-11S,12S-diol and DB[a,l]P-(-)-trans-11R,12R-diol. Glucuronidation assays with HEK293 cell lines overexpressing individual human UGT enzymes demonstrated that UGTs 1A1, 1A4, 1A7, 1A8, 1A9, 1A10, and 2B7 glucuronidated one or both DB[a,l]P-trans-11,12-diol enantiomers. Three glucuronide conjugates were observed in activity assays with UGTs 1A1 and 1A10, while two glucuronides were formed by UGTs 1A7, 1A8, and 1A9, and one glucuronide was made by UGT1A4 and UGT2B7. Enzyme kinetic analysis indicated that UGT1A9 was the most efficient UGT at forming both the (+)-DB[a,l]P-11-Gluc and (-)-DB[a,l]P-11-Gluc products, while UGTs 1A1 and 1A10 were the most efficient at forming the (+)-DB[a,l]P-12-Gluc product (as determined by k(cat)/K(M)). Incubations with human liver microsomes showed the formation of three diastereomeric glucuronide products: (+)-DB[a,l]P-11-Gluc, (+)-DB[a,l]P-12-Gluc, and (-)-DB[a,l]P-11-Gluc, with an average overall ratio of 31:32:37 in four liver specimens. Human bronchus and trachea tissue homogenates demonstrated glucuronidation activity against both DB[a,l]P-trans-11,12-diol enantiomers, with both tissues producing the (+)-DB[a,l]P-11-Gluc and (+)-DB[a,l]P-12-Gluc with little or no formation of (-)-DB[a,l]P-11-Gluc. These results indicate that multiple UGTs are involved in the stereospecific glucuronidation of DB[a,l]P-trans-11,12-diol in a pattern consistent with their expression in respiratory tract tissues and that glucuronidation may be an important first-line detoxification mechanism of DB[a,l]P metabolites.

Facile syntheses of O(2)-[4-(3-pyridyl-4-oxobut-1-yl]thymidine, the major adduct formed by tobacco specific nitrosamine 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) in vivo, and its site-specifically adducted oligodeoxynucleotides.

O(2)-[4-(3-Pyridyl)-4-oxobut-1-yl]thymidine (O(2)-POB-dThd) is the most persistent adduct detected in the lung and liver of rats treated with tobacco specific nitrosamines: N'-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and its metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL). It is an important biomarker to assess the human exposure to these carcinogens. The only synthetic method reported for O(2)-POB-dThd requires repeated HPLC purifications and could only be used to prepare an analytical standard due to very low yield (0.4%). We have developed for the first time a regioselective and efficient method for the total synthesis of O(2)-POB-dThd and its site-specifically adducted oligonucleotides. The main step in the synthesis of O(2)-POB-dThd was achieved by a novel method. The treatment of O(2)-5'-anhydrothymidine with the sodium salt of 4-(1,3-dithian-2-yl)-4-(3-pyridyl)butan-1-ol gave exclusively the O(2)-alkylated adduct, which was deprotected in one step to furnish the desired O(2)-POB-dThd in excellent yield. The product was characterized by NMR ((1)H and (13)C), high-resolution MS, and HPLC analysis. This work provided for the first time a reliable method for large scale total synthesis of O(2)-POB-dThd that allowed for solid state site-specifically adducted oligomer synthesis. The O(2)-POB-dThd was converted to its phosphoramidite and subsequently used for the synthesis of oligodeoxynucleotides by standard methods. The oligomers were characterized by MS and HPLC analysis. These oligomers will facilitate the elucidation of the mutagenic potential of the O(2)-POB-dThd adduct, which will provide further insight into the role of tobacco-specific nitrosamines in inducing cancers in smokers.

Incorporation of gemcitabine and cytarabine into DNA by DNA polymerase beta and ligase III/XRCC1.

1-Beta-D-arabinofuranosylcytosine (cytarabine, araC) and 2',2'-difluoro-2'-deoxycytidine (gemcitabine, dFdC), are effective cancer chemotherapeutic agents due to their ability to become incorporated into DNA and then subsequently inhibit DNA synthesis by replicative DNA polymerases. However, the impact of these 3'-modified nucleotides on the activity of specialized DNA polymerases has not been investigated. The role of polymerase beta and base excision repair may be of particular importance due to the increased oxidative stress in tumors, increased oxidative stress caused by chemotherapy treatment, and the variable amounts of polymerase beta in tumors. Here we directly investigate the incorporation of the 5'-triphosphorylated form of araC, dFdC, 2'-fluoro-2'-deoxycytidine (FdC), and cytidine into two nicked DNA substrates and the subsequent ligation. Opposite template dG, the relative k(pol)/K(d) for incorporation was dCTP > araCTP, dFdCTP >> rCTP. The relative k(pol)/K(d) for FdCTP depended on sequence. The effect on k(pol)/K(d) was due largely to changes in k(pol) with no differences in the affinity of the nucleoside triphosphates to the polymerase. Ligation efficiency by T4 ligase and ligase III/XRCC1 was largely unaffected by the nucleotide analogues. Our results show that BER is capable of incorporating araC and dFdC into the genome.

Identifying the features of purine dNTPs that allow accurate and efficient DNA replication by herpes simplex virus I DNA polymerase.

To accurately replicate its viral genome, the Herpes Simplex Virus 1 (HSV-1) DNA polymerase usually polymerizes the correct natural 2'-deoxy-5'-triphosphate (dNTP) opposite the template base being replicated. We employed a series of purine-dNTP analogues to determine the chemical features of the base necessary for the herpes polymerase to avoid polymerizing incorrect dNTPs. The enzyme uses N-3 to prevent misincorporation of purine dNTPs but does not require N-3 for correct polymerization. A free pair of electrons on N-1 also helps prevent misincorporation opposite A, C, and G and strongly drives polymerization opposite T. N6 contributes a small amount both for preventing misincorporation and for correct polymerization. Within the context of guanine in either the incoming dNTP or the template base being replicated, N2 prevents misincorporation opposite adenine but plays at most a minor role for incorporation opposite C. In contrast, adding N2 to the dNTPs of either adenine, purine, 6-chloropurine, or 1-deazapurine greatly enhances incorporation opposite C, likely via the formation of a hydrogen bond between N2 of the purine and O2 of the pyrimidine. Herpes polymerase is very sensitive to the structure of the base pair at the primer 3'-terminus since eliminating N-1, N-3, or N6 from a purine nucleotide at the primer 3'-terminus interfered with polymerization of the next two dNTPs. The biological and evolutionary implications of these data are discussed.

Discrimination between right and wrong purine dNTPs by DNA polymerase I from Bacillus stearothermophilus.

We used a series of dATP and dGTP analogues to determine how DNA polymerase I from Bacillus stearothermophilus (BF), a prototypical A family polymerase, uses N-1, N(2), N-3, and N(6) of purine dNTPs to differentiate between right and wrong nucleotide incorporation. Altering any of these nitrogens had two effects. First, it decreased the efficiency of correct incorporation of the resulting dNTP analogue, with the loss of N-1 and N-3 having the most severe effects. Second, it dramatically increased the rate of misincorporation of the resulting dNTP analogues, with alterations in either N-1 or N(6) having the most severe impacts. Adding N(2) to dNTPs containing the bases adenine and purine increased the degree of polymerization opposite T but also tremendously increased the degree of misincorporation opposite A, C, and G. Thus, BF uses N-1, N(2), N-3, and N(6) of purine dNTPs both as negative selectors to prevent misincorporation and as positive selectors to enhance correct incorporation. Comparing how BF discriminates between right and wrong dNTPs with both B family polymerases and low-fidelity polymerases indicates that BF has chosen a unique solution vis-a-vis these other enzymes and, therefore, that nature has evolved at least three mechanistically distinct solutions.

Modulations of benzo[a]pyrene-induced DNA adduct, cyclin D1 and PCNA in oral tissue by 1,4-phenylenebis(methylene)selenocyanate.

Tobacco smoking is an important cause of human oral squamous cell carcinoma (SCC). Tobacco smoke contains multiple carcinogens include polycyclic aromatic hydrocarbons typified by benzo[a]pyrene (B[a]P). Surgery is the conventional treatment approach for SCC, but it remains imperfect. However, chemoprevention is a plausible strategy and we had previously demonstrated that 1,4-phenylenebis(methylene)selenocyanate (p-XSC) significantly inhibited tongue tumors-induced by the synthetic 4-nitroquinoline-N-oxide (not present in tobacco smoke). In this study, we demonstrated that p-XSC is capable of inhibiting B[a]P-DNA adduct formation, cell proliferation, cyclin D1 expression in human oral cells in vitro. In addition, we showed that dietary p-XSC inhibits B[a]P-DNA adduct formation, cell proliferation and cyclin D1 protein expression in the mouse tongue in vivo. The results of this study are encouraging to further evaluate the chemopreventive efficacy of p-XSC initially against B[a]P-induced tongue tumors in mice and ultimately in the clinic.

Functional characterization of low-prevalence missense polymorphisms in the UDP-glucuronosyltransferase 1A9 gene.

The UDP-glucuronosyltransferase (UGT) 1A9 has been shown to play an important role in the detoxification of several carcinogens and clearance of anticancer and pain medications. The goal of the present study was to identify novel polymorphisms in UGT1A9 and characterize their effect on glucuronidation activity. The UGT1A9 gene was analyzed by direct sequencing of buccal cell genomic DNA from 90 healthy subjects. In addition to a previously identified single nucleotide polymorphism (SNP) at codon 33 resulting in an amino acid substitution (Met>Thr), two low-prevalence (<0.02) novel missense SNPs at codons 167 (Val>Ala) and 183 (Cys>Gly) were identified and are present in both white and African-American subjects. Glucuronidation activity assays using HEK293 cell lines overexpressing wild-type or variant UGT1A9 demonstrated that the UGT1A9(33Thr) and UGT1A9(183Gly) variants exhibited differential glucuronidation activities compared with wild-type UGT1A9, but this was substrate-dependent. The UGT1A9(167Ala) variant exhibited levels of activity similar to those of wild-type UGT1A9 for all substrates tested. Whereas the wild-type and UGT1A9(33Thr) and UGT1A9(167Ala) variants formed homodimers as determined by Western blot analysis of native polyacrylamide gels, the UGT1A9(183Gly) variant was incapable of homodimerization. These results suggest that several low-prevalence missense polymorphisms exist for UGT1A9 and that two of these (M33T and C183G) are functional. These results also suggest that although Cys183 is necessary for UGT1A9 homodimerization, the lack of capacity for UGT1A9 homodimerization is not sufficient to eliminate UGT1A9 activity.