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F Peter Guengerich

Publications and source records attributed to F Peter Guengerich.

At least 19 recordsLinked to original sources
PubMed2007

Multiple sequential steps involved in the binding of inhibitors to cytochrome P450 3A4.

Cytochrome P450 (P450) 3A4 is an extensively studied human enzyme involved in the metabolism of >50% of drugs. The mechanism of the observed homotropic and heterotropic cooperativity in P450 3A4-catalyzed oxidations is not well understood, and together with the cooperative behavior, a detailed understanding of interaction of drug inhibitors with P450 3A4 is important in predicting clinical drug-drug interactions. The interactions of P450 3A4 with several structurally diverse inhibitors were investigated using both kinetic and thermodynamic approaches to resolve the steps involved in binding of these ligands. The results of pre-steady-state absorbance and fluorescence experiments demonstrate that inhibitor binding is clearly a multistep process, even more complex than the binding of substrates. Based on spectrophotometric equilibrium binding titrations as well as isothermal titration calorimetry experiments, the stoichiometry of binding appears to be 1:1 in the concentration ranges studied. Using a sequential-mixing stopped-flow approach, we were also able to show that the observed multiphasic binding kinetics is the result of sequential events as opposed to the existence of multiple enzyme populations in dynamic equilibrium that interact with ligands at different rates. We propose a three-step minimal model for inhibitor binding, developed with kinetic simulations, consistent with our previously reported model for the binding of substrates, although it is possible that even more steps are involved.

Cytochrome P-450 CYP3A↗
PubMed2007

Effect of alterations of key active site residues in O6-alkylguanine-DNA Alkyltransferase on its ability to modulate the genotoxicity of 1,2-dibromoethane.

The production of mutations and the reduction in survival of cells treated with alpha,omega-dihaloalkanes is greatly enhanced by the presence of O6-alkylguanine-DNA alkyltransferase (AGT), a DNA repair protein that removes O6-alkylguanine adducts from DNA [Liu, L., Hachey, D. L., Valadez, G., Williams, K. M., Guengerich, F. P., Loktionova, N. A., Kanugula, S., and Pegg, A. E. (2004) J. Biol. Chem. 279, 4250-4259]. The effects of alterations to key residues in the active site of AGT were studied using AGTs with point mutations. It was found that mutants of AGT at positions Tyr114, Arg128, Pro140, Gly156, Gly160, and Tyr158 did not bring about the increase in genotoxicity of 1,2-dibromoethane seen with wild-type AGT, although these mutants, with the exception of those at Tyr114 and Arg128, are known to have sufficient AGT repair function to be able to protect cells from alkylating agents. The R128A mutant was able to react with 1,2-dibromoethane at the Cys145 acceptor site, but the resulting AGT-Cys145S-(CH2)2Br was much less able to produce a covalent adduct with DNA. This result is explained by the need for AGT to induce a structural change in the DNA "flipping" of a guanine nucleotide into the substrate binding pocket where Cys145 is located since the side chain of residue Arg128 plays a critical role in this reaction. Point mutations in AGT at the other sites (Y114A, P140K, and Y158H) reduced the ability of the protein to react with 1,2-dibromoethane as measured by the loss of activity. These results were confirmed by MS analysis of the tryptic peptide that contains the modified Cys145. There was no change in the stability of the AGT-Cys145S-(CH2)2Br intermediate formed in mutants Y158H and P140K. The reaction was studied in detail with mutant P140K using dihaloalkanes of different length; no effect of the mutations was seen with dibromomethane, but an enhanced difference was observed with 1,3-dibromopropane and 1,5-dibromopentane. These results show that even slight alterations in the active site pocket of AGT that do not prevent its ability to protect cells from alkylating agents can block the paradoxical enhancement of the genotoxicity of the larger alpha,omega-dihaloalkanes by reducing the reaction with Cys145.

Binding Sites↗
PubMed2007

Formation of the indigo precursor indican in genetically engineered tobacco plants and cell cultures.

The production of the blue dye indigo in plants has been assumed to be a possible route to the introduction of novel coloration into flowers or fibres. As the human cytochrome P450 mono-oxygenase 2A6 (CYP2A6) can form indigo in bacterial cultures, we investigated whether the expression of the corresponding cDNA in transgenic plants could lead to indigo formation. In a first attempt, we generated tobacco cell suspension cultures expressing the cDNA encoding human CYP2A6. Supplementation of the medium with indole led to the generation of indican (3-hydroxyindole-beta-d-glucoside), a metabolite usually exclusively present in indigoferous dye plants. Hence, the recombinant CYP2A6 converted indole to the reactive metabolite 3-hydroxyindole (indoxyl), whereas rapid glucosylation is obviously conducted by ubiquitous plant glucosyl transferases (GTs). Interestingly, of nine additionally tested plant cell suspension cultures from various plant families, five were also capable of the formation of indican after indole supplementation, although this metabolism was more pronounced in transgenic tobacco cell suspension cultures expressing CYP2A6 cDNA. To evaluate whether indican or even indigo could be produced in whole plants, we generated transgenic tobacco plants harbouring active CYP2A6 together with an indole synthase (BX1) from maize. The genetically engineered tobacco plants accumulated indican, but did not develop a blue coloration. Although the de novo formation of indican in transgenic tobacco plants hampered indigo formation, it supports the contention that biosynthetic pathways can be efficiently mimicked by metabolic engineering.

Aryl Hydrocarbon Hydroxylases↗
PubMed2006

Sulfolobus solfataricus DNA polymerase Dpo4 is partially inhibited by "wobble" pairing between O6-methylguanine and cytosine, but accurate bypass is preferred.

We examined the effect of a single O6-methylguanine (O6-MeG) template residue on catalysis by a model Y family polymerase, Dpo4 from Sulfolobus solfataricus. Mass spectral analysis of Dpo4-catalyzed extension products revealed that the enzyme accurately bypasses O6-MeG, with C being the major product (approximately 70%) and T or A being the minor species (approximately 20% or approximately 10%, respectively), consistent with steady-state kinetic parameters. Transient-state kinetic experiments revealed that kpol, the maximum forward rate constant describing polymerization, for dCTP incorporation opposite O6-MeG was approximately 6-fold slower than observed for unmodified G, and no measurable product was observed for dTTP incorporation in the pre-steady state. The lack of any structural information regarding how O6-MeG paired in a polymerase active site led us to perform x-ray crystallographic studies, which show that "wobble" pairing occurs between C and O6-MeG. A structure containing T opposite O6-MeG was solved, but much of the ribose and pyrimidine base density was disordered, in accordance with a much higher Km,dTTP that drives the difference in efficiency between C and T incorporation. The more stabilized C:O6-MeG pairing reinforces the importance of hydrogen bonding with respect to nucleotide selection within a geometrically tolerant polymerase active site.

Binding Sites↗
PubMed2006

Activation of aminophenylnorharman, aminomethylphenylnorharman and aminophenylharman to genotoxic metabolites by human N-acetyltransferases and cytochrome P450 enzymes expressed in Salmonella typhimurium umu tester strains.

Norharman (9H-pyrido[3,4-b]indole) and harman (1-methyl-9H-pyrido[3,4-b]indole) contained in cigarette smoke and cooked foodstuffs, are non-mutagenic to Salmonella strains, but show co-mutagenicity with S9 mixture in the presence of aniline or o-toluidine. The resulting 9-(4'-aminophenyl)-9H-pyrido[3,4-b]indole (aminophenylnorharman, APNH), 9-(4'-amino-3'-methylphenyl)-9H-pyrido[3,4-b]indole (aminomethylphenylnorharman, AMPNH) and 9-(4'-aminophenyl)-1-methyl-9H-pyrido[3,4-b]indole (aminophenylharman, APH) are produced by coupling of norharman and aniline, norharman and o-toluidine, and harman and aniline in the presence of S9 mixture, respectively. To clarify the role of human cytochrome P450 (P450) and N-acetyltransferase (NAT) enzymes in the metabolic activation of APNH, AMPNH and APH, we determined the genotoxicity of these coupling chemicals using a variety of umu tester strains established in our laboratories. APNH, AMPNH and APH induced umuC gene expression more strongly in a bacterial O-acetyltransferase-overproducing strain than the parent strain. These chemicals were also found to induce umuC gene expression in NAT2-overexpressing strain at much higher rate than the NAT1-overexpressing strain. Among seven OY strains expressing human P450s and NADPH-P450 reductase used, the genotoxicity of APNH, AMPNH and APH was detected in OY1002/1A2 strain, OY1002/1A1 and OY1002/1A2 strains, and in OY1002/1A2 strain, respectively. From these results, it is concluded that APNH, AMPNH and APH are mainly bioactivated by P450 1A2 and NAT2, followed by NAT1 enzymes. P450 1A1 was also found to activate AMPNH at relatively slower rates.

Aniline Compounds↗
PubMed2006

Translesion synthesis across O6-alkylguanine DNA adducts by recombinant human DNA polymerases.

Previous studies have shown that replicative bacterial and viral DNA polymerases are able to bypass the mutagenic lesions O(6)-methyl and -benzyl (Bz) G. Recombinant human polymerase (pol) delta also copied past these two lesions but was totally blocked by O(6)-[4-oxo-4-(3-pyridyl)butyl] (Pob)G, an important mutagenic lesion formed following metabolic activation of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. The human translesion pols iota and kappa produced mainly only 1-base incorporation opposite O(6)-MeG and O(6)-BzG and had very low activity in copying O(6)-PobG. Human pol eta copied past all three adducts. Steady-state kinetic analysis showed similar efficiencies of insertion opposite the O(6)-alkylG adducts for dCTP and dTTP with pol eta and kappa; pol iota showed a strong preference for dTTP. pol eta, iota, and kappa showed pre-steady-state kinetic bursts for dCTP incorporation opposite G and O(6)-MeG but little, if any, for O(6)-BzG or O(6)-PobG. Analysis of the pol eta O(6)-PobG products indicated that the insertion of G was opposite the base (C) 5' of the adduct, but this product was not extended. Mass spectrometry analysis of all of the pol eta primer extension products indicated multiple components, mainly with C or T inserted opposite O(6)-alkylG but with no deletions in the cases of O(6)-MeG and O(6)-PobG. With pol eta and O(6)-BzG, products were also obtained with -1 and -2 deletions and also with A inserted (opposite O(6)-BzG). The results with pol eta may be relevant to some mutations previously reported with O(6)-alkylG adducts in mammalian cells.

Base Sequence↗
PubMed2006

Biochemical basis of genotoxicity of heterocyclic arylamine food mutagens: Human DNA polymerase eta selectively produces a two-base deletion in copying the N2-guanyl adduct of 2-amino-3-methylimidazo[4,5-f]quinoline but not the C8 adduct at the NarI G3 site.

Heterocyclic arylamines are highly mutagenic and cause tumors in animal models. The mutagenicity is attributed to the C8- and N2-G adducts, the latter of which accumulates due to slower repair. The C8- and N 2-G adducts derived from 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) were placed at the G1 and G3 sites of the NarI sequence, in which the G3 site is an established hot spot for frameshift mutation with the model arylamine derivative 2-acetylaminofluorene but G1 is not. Human DNA polymerase (pol) eta extended primers beyond template G-IQ adducts better than did pol kappa and much better than pol iota or delta. In 1-base incorporation studies, pol eta inserted C and A, pol iota inserted T, and pol kappa inserted G. Steady-state kinetic parameters were measured for these dNTPs opposite the C8- and N 2-IQ adducts at both sites, being most favorable for pol eta. Mass spectrometry of pol eta extension products revealed a single major product in each of four cases; with the G1 and G3 C8-IQ adducts, incorporation was largely error-free. With the G3 N 2-IQ adduct, a -2 deletion occurred at the site of the adduct. With the G1 N 2-IQ adduct, the product was error-free at the site opposite the base and then stalled. Thus, the pol eta products yielded frame-shifts with the N 2 but not the C8 IQ adducts. We show a role for pol eta and the complexity of different chemical adducts of IQ, DNA position, and DNA polymerases.

Biochemistry↗
PubMed2006

Translesion synthesis across bulky N2-alkyl guanine DNA adducts by human DNA polymerase kappa.

DNA polymerase (pol) kappa is one of the so-called translesion polymerases involved in replication past DNA lesions. Bypass events have been studied with a number of chemical modifications with human pol kappa, and the conclusion has been presented, based on limited quantitative data, that the enzyme is ineffective at incorporating opposite DNA damage but proficient at extending beyond bases paired with the damage. Purified recombinant full-length human pol kappa was studied with a series of eight N(2)-guanyl adducts (in oligonucleotides) ranging in size from methyl- to -CH(2)(6-benzo[a]pyrenyl) (BP). Steady-state kinetic parameters (catalytic specificity, k(cat)/K(m)) were similar for insertion of dCTP opposite the lesions and for extension beyond the N(2)-adduct G:C pairs. Mispairing of dGTP and dTTP was similar and occurred with k(cat)/K(m) values approximately 10(-3) less than for dCTP with all adducts; a similar differential was found for extension beyond a paired adduct. Pre-steady-state kinetic analysis showed moderately rapid burst kinetics for dCTP incorporations, even opposite the bulky methyl(9-anthracenyl)- and BPG adducts (k(p) 5.9-10.3 s(-1)). The rapid bursts were abolished opposite BPG when alpha-thio-dCTP was used instead of dCTP, implying rate-limiting phosphodiester bond formation. Comparisons are made with similar studies done with human pols eta and iota; pol kappa is the most resistant to N(2)-bulk and the most quantitatively efficient of these in catalyzing dCTP incorporation opposite bulky guanine N(2)-adducts, particularly the largest (N(2)-BPG).

Baculoviridae↗
PubMed2006

Chemical reactivity as a tool to study carcinogenicity: reaction between chloroethylene oxide and guanine.

Chloroethylene oxide, an ultimate carcinogen of vinyl chloride, reacts with DNA giving rise to 7-(2-oxyethyl)guanine adduct in a nearly quantitative yield. This reaction represents an initial step of carcinogenesis associated with vinyl chloride. From experimental data for this reaction we calculated the second-order rate constant of 0.049 s(-1) M(-1), which corresponds to the activation free energy of 19.5 kcal/mol. We also performed a series of medium high ab initio and density functional theory simulations. Effects of hydration were considered in the framework of the Langevine dipoles solvation model and the solvent reaction field method of Tomasi and co-workers. In silico calculated activation free energies are in a good agreement with the experimental value. This fact presents strong evidence in favor of the validity of the proposed reaction mechanism and points to the applicability of quantum-chemical methods to studies of other reactions associated with carcinogenesis. Insignificant stereoselectivity of the studied reaction was also predicted.

Carcinogens↗
PubMed2006

Calcium is a cofactor of polymerization but inhibits pyrophosphorolysis by the Sulfolobus solfataricus DNA polymerase Dpo4.

Y-Family DNA polymerase IV (Dpo4) from Sulfolobus solfataricus serves as a model system for eukaryotic translesion polymerases, and three-dimensional structures of its complexes with native and adducted DNA have been analyzed in considerable detail. Dpo4 lacks a proofreading exonuclease activity common in replicative polymerases but uses pyrophosphorolysis to reduce the likelihood of incorporation of an incorrect base. Mg(2+) is a cofactor for both the polymerase and pyrophosphorolysis activities. Despite the fact that all crystal structures of Dpo4 have been obtained in the presence of Ca(2+), the consequences of replacing Mg(2+) with Ca(2+) for Dpo4 activity have not been investigated to date. We show here that Ca(2+) (but not Ba(2+), Co(2+), Cu(2+), Ni(2+), or Zn(2+)) is a cofactor for Dpo4-catalyzed polymerization with both native and 8-oxoG-containing DNA templates. Both dNTP and ddNTP are substrates of the polymerase in the presence of either Mg(2+) or Ca(2+). Conversely, no pyrophosphorolysis occurs in the presence of Ca(2+), although the positions of the two catalytic metal ions at the active site appear to be very similar in mixed Mg(2+)/Ca(2+)- and Ca(2+)-form Dpo4 crystals.

Base Sequence↗
PubMed2006

Recombinant enzymes overexpressed in bacteria show broad catalytic specificity of human cytochrome P450 2W1 and limited activity of human cytochrome P450 2S1.

Human cytochromes P450 2S1 and 2W1 have received only limited attention with regard to characterization of function. Both cytochromes P450 have been reported to be overexpressed in human tumors, and cytochrome P450 2S1 is induced by carcinogenic polycyclic hydrocarbons. We report methods for high-level expression and purification of both cytochromes P450 from Escherichia coli, with the goal of establishing function. The level of expression of human cytochrome P450 2W1 achieved using codon optimization for E. coli was 1800 nmol of cytochrome P450 per liter of culture, the highest level achieved in this laboratory to date. Assays with a number of the typical cytochrome P450 substrates showed no detectable activity, including some for which qualitative reports have appeared in the literature. Cytochrome P450 2W1 catalyzed benzphetamine N-demethylation (k(cat), 3.8/min) and arachidonic acid oxidation, albeit at a very low rate (approximately 0.05/min). In a umu genotoxicity screen, cytochrome P450 2W1 catalyzed the activation of several procarcinogens, particularly polycyclic hydrocarbon diols, but cytochrome P450 2S1 did not. The bioactivation of procarcinogens by cytochrome P450 2W1 may be of significance in the context of reports of preferential expression of the enzyme in tumors, in that activation of procarcinogens could lead to the accumulation of mutations and enhance the carcinogenic process.

Amino Acid Sequence↗
PubMed2006

Cytochrome P450s and other enzymes in drug metabolism and toxicity.

The cytochrome P450 (P450) enzymes are the major catalysts involved in the metabolism of drugs. Bioavailability and toxicity are 2 of the most common barriers in drug development today, and P450 and the conjugation enzymes can influence these effects. The toxicity of drugs can be considered in 5 contexts: on-target toxicity, hypersensitivity and immunological reactions, off-target pharmacology, bioactivation to reactive intermediates, and idiosyncratic drug reactions. The chemistry of bioactivation is reasonably well understood, but the mechanisms underlying biological responses are not. In the article we consider what fraction of drug toxicity actually involves metabolism, and we examine how species and human interindividual variations affect pharmacokinetics and toxicity.

Animals↗
PubMed2006

Kinetic evidence for inefficient and error-prone bypass across bulky N2-guanine DNA adducts by human DNA polymerase iota.

DNA polymerase (pol) iota has been proposed to be involved in translesion synthesis past minor groove DNA adducts via Hoogsteen base pairing. The N2 position of G, located in minor groove side of duplex DNA, is a major site for DNA modification by various carcinogens. Oligonucleotides with varying adduct size at G N2 were analyzed for bypass ability and fidelity with human pol iota. Pol iota effectively bypassed N2-methyl (Me)G and N2-ethyl(Et)G, partially bypassed N2-isobutyl(Ib)G and N2-benzylG, and was blocked at N2-CH2(2-naphthyl)G (N2-NaphG), N2-CH2(9-anthracenyl)G (N2-AnthG), and N2-CH2(6-benzo[a]pyrenyl)G. Steady-state kinetic analysis showed decreases of kcat/Km for dCTP insertion opposite N2-G adducts according to size, with a maximal decrease opposite N2-AnthG (61-fold). dTTP misinsertion frequency opposite template G was increased 3-11-fold opposite adducts (highest with N2-NaphG), indicating the additive effect of bulk (or possibly hydrophobicity) on T misincorporation. N2-IbG, N2-NaphG, and N2-AnthG also decreased the pre-steady-state kinetic burst rate compared with unmodified G. High kinetic thio effects (S(p)-2'-deoxycytidine 5'-O-(1-thiotriphosphate)) opposite N2-EtG and N2-AnthG (but not G) suggest that the chemistry step is largely interfered with by adducts. Severe inhibition of polymerization opposite N2,N2-diMeG compared with N2-EtG by pol eta but not by pol iota is consistent with Hoogsteen base pairing by pol iota. Thus, polymerization by pol iota is severely inhibited by a bulky group at G N2 despite an advantageous mode of Hoogsteen base pairing; pol iota may play a limited role in translesion synthesis on bulky N2-G adducts in cells.

Baculoviridae↗
PubMed2006

Kinetics and thermodynamics of ligand binding by cytochrome P450 3A4.

Cytochrome P450 (P450) 3A4, the major catalyst involved in human drug oxidation, displays substrate- and reaction-dependent homotropic and heterotropic cooperative behavior. Although several models have been proposed, these mainly rely on steady-state kinetics and do not provide information on the contribution of the individual steps of P450 catalytic cycle to the observed cooperativity. In this work, we focused on the kinetics of substrate binding, and the fluorescent properties of bromocriptine and alpha-naphthoflavone allowed analysis of an initial ligand-P450 3A4 interaction that does not cause a perturbation of the heme spectrum. The binding stoichiometry for bromocriptine was determined to be unity using isothermal titration calorimetry and equilibrium dialysis methods, suggesting that the ligand bound to the peripheral site during the initial encounter dissociates subsequently. A three-step substrate binding model is proposed, based on absorbance and fluorescence stopped-flow kinetic data and equilibrium binding data obtained with bromocriptine, and evaluated using kinetic modeling. The results are consistent with the substrate molecule binding at a site peripheral to the active site and subsequently moving toward the active site to bind to the heme and resulting in a low to high spin iron shift. The last step is attributed to a conformational change in the enzyme active site. The later steps of binding were shown to have rate constants comparable with the subsequent steps of the catalytic cycle. The P450 3A4 binding process is more complex than a two-state system, and the overlap of rates of some of the events with subsequent steps is proposed to underlie the observed cooperativity.

Bromocriptine↗
PubMed2006

Inhibition of human cytochrome P450 1A1-, 1A2-, and 1B1-mediated activation of procarcinogens to genotoxic metabolites by polycyclic aromatic hydrocarbons.

Many chemicals in the environment can cause cancer, and polycyclic aromatic hydrocarbons (PAHs) are among the most ubiquitous. Cancer risk assessments require consideration of these in complex mixtures. PAHs require metabolic activation by cytochrome P450 (P450) enzymes, primarily 1A1, 1A2, and 1B1. We determined if individual PAHs and other procarcinogens affect the activities of human P450s 1A1, 1A2, and 1B1 by measuring 7-ethoxyresorufin O-deethylation (EROD) activity and metabolic activation of PAH dihydrodiols and 2-amino-3,5-dimethylimidazo[4,5-f]quinoline (MeIQ) to genotoxic metabolites in a Salmonella typhimurium NM2009 system. Of 23 PAHs examined, benz[a]anthracene (B[a]A), benzo[b]fluoranthene, and 5-methylchrysene were the most potent inhibitors of P450 1A2- and 1B1-catalyzed EROD activity, with IC50 values <10 nM. Other PAHs, e.g., dibenz[a,c]anthracene, dibenz[a,h]anthracene, dibenz[a,j]acridine, and 3-methylcholanthrene, rather selectively inhibited P450 1B1, with IC50 values <15 nM. Benzo[a]pyrene (B[a]P) and nine other PAHs also inhibited P450 1A2 as well as 1B1 with IC50 values <150 nM. Parent PAH compounds were generally more potent than 10 dihydrodiol metabolites of PAHs and 3- and 9-hydroxy B[a]P in inhibiting EROD activity. In addition, we found that three selected PAHs (5-methylchrysene, B[a]P, and B[a]A) inhibited metabolic activation of 5-methylchrysene-1,2-diol, (+/-)-B[a]P-7,8-diol, dibenzo[a,l]pyrene-11,12-diol, and MeIQ to genotoxic metabolites catalyzed by P450s 1A1, 1B1, and 1A2, respectively, in S. typhimurium NM2009. Thus, individual PAHs may affect their own and metabolism of other carcinogens catalyzed by P450 1A1, 1A2, and 1B1, and these phenomena cause alteration in their ability to transform cells when single or complex PAH mixtures are ingested by mammals, influencing risk assessment.

Aryl Hydrocarbon Hydroxylases↗
PubMed2006

Translesion synthesis across 1,N2-ethenoguanine by human DNA polymerases.

1,N(2)-Etheno(epsilon)guanine (epsilon) is formed in DNA as a result of exposure to certain vinyl monomers (e.g., vinyl chloride) or from lipid peroxidation. This lesion has been shown to be mutagenic in bacteria and mammalian cells. 1,N(2)-epsilon-G has been shown to block several model replicative DNA polymerases (pols), with limited bypass. Recently, an archebacterial DNA pol, Sulfolobus solfataricus Dpo4, has been shown to copy past 1,N(2)-epsilon-G. In this study, we examined the abilities of recombinant, full-length human pol delta and three human translesion DNA pols to copy past 1,N(2)-epsilon-G. The replicative pol, pol delta, was completely blocked. Pols iota and kappa showed similar rates of incorporation of dTTP and dCTP. Pol eta was clearly the most active of these pols in copying past 1,N(2)-epsilon-G, incorporating in the order dGTP > dATP > dCTP, regardless of whether the base 5' of 1,N(2)-epsilon-G in the template was C or T. Pol eta also had the highest error frequency opposite 1,N(2)-epsilon-G. Analysis of the extended products of the pol eta reactions by mass spectrometry indicated only two products, both of which had G incorporated opposite 1,N(2)-epsilon-G and all other base pairing being normal (i.e., G:C and A:T). One-half of the products contained an additional A at the 3'-end, presumably arising from a noninformational blunt end addition or possibly a slipped insertion mechanism at the end of the primer-template replication process. In summary, the most efficient of the four human DNA pols was pol eta, which appeared to insert G opposite 1,N(2)-epsilon-G and then copy correctly. This pattern differs with the same oligonucleotide sequences and 1,N(2)-epsilon-G observed using Dpo4, emphasizing the importance of pols in mutagenesis events.

DNA Damage↗