Basis of Miscoding of the DNA Adduct <i>N</i>²,3-Ethenoguanine by Human Y-family DNA Polymerases

Zhao, Linlin,Pence, Matthew G.,Christov, Plamen P.,Wawrzak, Zdzislaw,Choi, Jeong-Yun,Rizzo, Carmelo J.,Egli, Martin,Guengerich, F. Peter American Society for Biochemistry and Molecular Bi 2012 The Journal of biological chemistry Vol.287 No.42

<P><I>N</I><SUP>2</SUP>,3-Ethenoguanine (<I>N</I><SUP>2</SUP>,3-ϵG) is one of the exocyclic DNA adducts produced by endogenous processes (<I>e.g.</I> lipid peroxidation) and exposure to bioactivated vinyl monomers such as vinyl chloride, which is a known human carcinogen. Existing studies exploring the miscoding potential of this lesion are quite indirect because of the lability of the glycosidic bond. We utilized a 2′-fluoro isostere approach to stabilize this lesion and synthesized oligonucleotides containing 2′-fluoro-<I>N</I><SUP>2</SUP>,3-ϵ-2′-deoxyarabinoguanosine to investigate the miscoding potential of <I>N</I><SUP>2</SUP>,3-ϵG by Y-family human DNA polymerases (pols). In primer extension assays, pol η and pol κ replicated through <I>N</I><SUP>2</SUP>,3-ϵG, whereas pol ι and REV1 yielded only 1-base incorporation. Steady-state kinetics revealed that dCTP incorporation is preferred opposite <I>N</I><SUP>2</SUP>,3-ϵG with relative efficiencies in the order of pol κ > REV1 > pol η ≈ pol ι, and dTTP misincorporation is the major miscoding event by all four Y-family human DNA pols. Pol ι had the highest dTTP misincorporation frequency (0.71) followed by pol η (0.63). REV1 misincorporated dTTP and dGTP with much lower frequencies. Crystal structures of pol ι with <I>N</I><SUP>2</SUP>,3-ϵG paired to dCTP and dTTP revealed Hoogsteen-like base pairing mechanisms. Two hydrogen bonds were observed in the <I>N</I><SUP>2</SUP>,3-ϵG:dCTP base pair, whereas only one appears to be present in the case of the <I>N</I><SUP>2</SUP>,3-ϵG:dTTP pair. Base pairing mechanisms derived from the crystal structures explain the slightly favored dCTP insertion for pol ι in steady-state kinetic analysis. Taken together, these results provide a basis for the mutagenic potential of <I>N</I><SUP>2</SUP>,3-ϵG.</P>

Effects of Twelve Germline Missense Variations on DNA Lesion and G-Quadruplex Bypass Activities of Human DNA Polymerase REV1

Yeom, Mina,Kim, In-Hyeok,Kim, Jae-Kwon,Kang, KyeongJin,Eoff, Robert L.,Guengerich, F. Peter,Choi, Jeong-Yun American Chemical Society 2016 Chemical research in toxicology Vol.29 No.3

<P>The Y-family DNA polymerase REV1 is involved in replicative bypass of damaged DNA and G-quadruplex (G4) DNA. In addition to a scaffolding role in the replicative bypass, REV1 acts in a catalytic role as a deoxycytidyl transferase opposite some replication stall sites, e.g., apurinic/apyrimidinic (AP) sites, N2-guanyl lesions, and G4 sites. We characterized the biochemical properties of 12 reported germline missense variants of human REV1, including the N373S variant associated with high risk of cervical cancer, using the recombinant REV1 (residues 330-833) proteins and DNA templates containing a G, AP site, N-2-CH2(2-naphthyl)G (N-2-NaphG), or G4. In steady-state kinetic analyses, the F427L, R434Q, M656V, D700N, R704Q, and P831L variants displayed 2- to 8-fold decreases in k(cat)/K-m for dCTP insertion opposite all four templates, compared to that of wild-type, while the N373S, M407L, and N497S showed 2- to 3-fold increases with all four and the former three or two templates, respectively. The F427L, R434Q, M656V, and R704Q variants also had 2- to 3-fold lower binding affinities to DNA substrates containing G, an AP site, and/or N-2-NaphG than wild-type. Distinctively, the N373S variant had a 3-fold higher binding affinity to G4 DNA than the wild-type, as well as a 2-fold higher catalytic activity opposite the first tetrad G, suggesting a facilitating effect of this variation on replication of G4 DNA sequences in certain human papillomavirus genomes. Our results suggest that the catalytic function of REV1 is moderately or slightly altered by at least nine genetic variations, and the G4 DNA processing function of REV1 is slightly enhanced by the N373S variation, which might provide the possibility that certain germline missense REV1 variations affect the individual susceptibility to carcinogenesis by modifying the capability of REV1 for replicative bypass past DNA lesions and G4 motifs derived from chemical and viral carcinogens.</P>

Inhibition of Cytochrome P450 Enzymes by Drugs―Molecular Basis and Practical Applications

( F. Peter Guengerich ) 한국응용약물학회 2022 Biomolecules & Therapeutics(구 응용약물학회지) Vol.30 No.1

Drug-drug interactions are a major cause of hospitalization and deaths related to drug use. A large fraction of these is due to inhibition of enzymes involved in drug metabolism and transport, particularly cytochrome P450 (P450) enzymes. Understanding basic mechanisms of enzyme inhibition is important, particularly in terms of reversibility and the use of the appropriate parameters. In addition to drug-drug interactions, issues have involved interactions of drugs with foods and natural products related to P450 enzymes. Predicting drug-drug interactions is a major effort in drug development in the pharmaceutical industry and regulatory agencies. With appropriate in vitro experiments, it is possible to stratify clinical drug-drug interaction studies. A better understanding of drug interactions and training of physicians and pharmacists has developed. Finally, some P450s have been the targets of drugs in some cancers and other disease states.

Activation of Dihaloalkanes by Thiol-dependent Mechanisms

Guengerich, F. Peter Korean Society for Biochemistry and Molecular Biol 2003 Journal of biochemistry and molecular biology Vol.36 No.1

Dihaloalkanes constitute an important group of chemicals because of their widespread use in industry and agriculture and their potential for causing toxicity and cancer. Chronic toxic effects are considered to depend upon bioactivation, either by oxidation or thiol conjugation. Considerable evidence links genotoxicity and cancer with glutathione conjugations reactions, and some aspects of the mechanisms have been clarified with 1,2-dihaloalkanes and dihalomethanes. Recently the DNA repair protein $O^6$-alkylguanine transferase has been shown to produce cytotoxicity and genotoxicity by mans of a thiol-dependent process with similarities to the glutathione reactions.

Biochemical Characterization of Eight Genetic Variants of Human DNA Polymerase κ Involved in Error-Free Bypass across Bulky <i>N</i>²-Guanyl DNA Adducts

Song, Insil,Kim, Eun-Jin,Kim, In-Hyeok,Park, Eun-Mi,Lee, Kyung Eun,Shin, Joo-Ho,Guengerich, F. Peter,Choi, Jeong-Yun American Chemical Society 2014 Chemical research in toxicology Vol.27 No.5

<P>DNA polymerase (pol) κ, one of the Y-family polymerases, has been shown to function in error-free translesion DNA synthesis (TLS) opposite the bulky <I>N</I><SUP>2</SUP>-guanyl DNA lesions induced by many carcinogens such as polycyclic aromatic hydrocarbons. We analyzed the biochemical properties of eight reported human pol κ variants positioned in the polymerase core domain, using the recombinant pol κ (residues 1–526) protein and the DNA template containing an <I>N</I><SUP>2</SUP>-CH<SUB>2</SUB>(9-anthracenyl)G (<I>N</I><SUP>2</SUP>-AnthG). The truncation R219X was devoid of polymerase activity, and the E419G and Y432S variants showed much lower polymerase activity than wild-type pol κ. In steady-state kinetic analyses, E419G and Y432S displayed 20- to 34-fold decreases in <I>k</I><SUB>cat</SUB>/<I>K</I><SUB>m</SUB> for dCTP insertion opposite G and <I>N</I><SUP>2</SUP>-AnthG compared to that of wild-type pol κ. The L21F, I39T, and D189G variants, as well as E419G and Y432S, displayed 6- to 22-fold decreases in <I>k</I><SUB>cat</SUB>/<I>K</I><SUB>m</SUB> for next-base extension from C paired with <I>N</I><SUP>2</SUP>-AnthG, compared to that of wild-type pol κ. The defective Y432S variant had 4- to 5-fold lower DNA-binding affinity than wild-type, while a slightly more efficient S423R variant possessed 2- to 3-fold higher DNA-binding affinity. These results suggest that R219X abolishes and the E419G, Y432S, L21F, I39T, and D189G variations substantially impair the TLS ability of pol κ opposite bulky <I>N</I><SUP>2</SUP>-G lesions in the insertion step opposite the lesion and/or the subsequent extension step, raising the possibility that certain nonsynonymous pol κ genetic variations translate into individual differences in susceptibility to genotoxic carcinogens.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/crtoec/2014/crtoec.2014.27.issue-5/tx500072m/production/images/medium/tx-2014-00072m_0006.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/tx500072m'>ACS Electronic Supporting Info</A></P>

Inhibition of 7-Alkoxyresorufin O-Dealkylation Activities of Recombinant Human CYP1A1 and CYP1B1 by Resveratrol

Mi-Sook Dong,Suk-Kyung Chang,Hyun-Jung Kim,Elizabeth M. J. Gillam,F. Peter Guengerich,Young In Park 한국환경성돌연변이발암원학회 2002 한국환경성돌연변이·발암원학회지 Vol.22 No.3

Resveratrol is known to have potent cancer chemopreventive activity against tumorigenesis caused by 7,12-dimethylbenz[α]anthracene (DMBA) which is known to be oxidized to reactive products by cytochrome P450 1B1 (CYP1B1). The effects of resveratrol on the activity of recombinant human P450 1 family enzymes, expressed in Escherichia coli membranes with human NADPH-P450 reductase, were determined by measuring alkoxyresorufin O-dealkylation activity, e.g., ethoxyresorufin O-deethylation (EROD) CYP1A1, methoxyresorufin O-demethylation (MROD), CYP1A2, benzyloxyresorufin-O-debenzylation (BROD), CYP1B1. Resveratrol<br/> inhibited CYP1B1 and CYP1A1 activities in a dose-dependent manner with IC50 values of 59 and 10 μM for EROD activity and 1.8 and 30 μM for BROD activity, respectively. Resveratrol had only weak inhibitory effect on CYP1A2 activity (IC50 values of 0.44 mM for EROD and >2 mM for MROD). Furthermore, resveratrol did not affect NADPH-P450 reductase activity significantly. Resveratrol inhibited the CYP1B1-dependent EROD activity with a Ki of 28 μM in a non-competitive type manner. These results suggest that resveratrol-derived inhibition of CYP1B1 and CYP1A1 activities may contribute to the suppression of DMBA inducible tumorigenesis observed in extrahepatic tissues.

Cytochrome P450 2A6 and other human P450 enzymes in the oxidation of flavone and flavanone

Kakimoto, Kensaku,Murayama, Norie,Takenaka, Shigeo,Nagayoshi, Haruna,Lim, Young-Ran,Kim, Vitchan,Kim, Donghak,Yamazaki, Hiroshi,Komori, Masayuki,Guengerich, F. Peter,Shimada, Tsutomu Taylor & Francis 2019 Xenobiotica Vol.49 No.2

Molecular Cloning and Expression of Fusion Proteins Containing Human Cytochrome P450 3As and Rat NADPH-P450 Reductase in Escherichia coli

Chun, Young-Jin,Guengerich, F-Peter Korean Society of ToxicologyKorea Environmental Mu 2002 Toxicological Research Vol.18 No.3

Cytochrome P450 3As such as 3A4 and 3A5 metabolize a wide range of pharmaceutical compounds. The vectors for the expression of fusion protein containing an N-terminal human P450 3A4 or P450 3A5 sequences and a C-terminal rat NADPH-cytochrome P450 reductase moiety were constructed. These plasmids were used to express the fusion protein in Escherichia coli DH5$\alpha$ cells. High levels of expression were achieved (100~200 nmol/liter) and the expressed fusion protein in E. coli membranes were catalytically active for nifedipine oxidation, a typical enzymatic activity of P450 3A4. The NADPH-P450 reductase activities of these fusion protein were also determined by measuring reduction of cytochrome c. To fine a specific Inhibitor of P450 3A4 from naturally occurring chemicals, a series of isothiocyanate compounds were evaluated for the inhibitory activity of P450 using the fusion proteins in E. coli membranes. Of the five isothiocyanates (phenethyl isothiocyanate, phenyl isothiocyanate, benzol isothiocyanate, benzoyl isothiocyanate and cyclohexyl isothiocyanate) tested, benzoyl isothiocyanate showed a strong inhibition of P450 3A4 with an $IC_{50}$value of 2.8 $\mu\textrm{M}$. Our results indicate that the self-sufficient fusion protein will be very useful tool to study the drug metabolism and benzyl isothiocyanate may be valuable for characterizing the enzymatic properties of P450 3A4.

OXIDATION OF THE ANGIOTENSIN Ⅱ RECEPTOR ANTAGONIST LOSARTAN (DuP 753) IN HUMAN LIVER MICROSOMES : Role of Cytochrome P4503A(4) in Formation of the Active Metabolite EXP3174

YUN, CHUL-HO,LEE, HYE SUK,LEE, HEEYONG,RHO, JUNG KOO,JEONG, HYE GWANG,F. PETER GUENGERICH 충남대학교 기초과학연구소 1995 연구논문집 Vol.15 No.-

The oxidative metabolism of losartan (DuP 753), a novel angiotensin Ⅱ receptor antagonist, by human liver microsomes and purified cytochrome P450(P450) enzymes, was studied. The primary route of metabolism of losartan is by oxidation of the C_5-hydroxymethyl to the carboxylic acid (EXP3174), which is and active metabolite of losartan. When microsomes prepared from different human liver samples were compared, EXP3174 formation activity was well correlated(r^2=0.93) with nifedipine oxidation (a maker of P4504A4), but not with markers for other human liver P450s. Microsomal oxidation of losartan to EXP3174 was markedly inhibited by gestodene and ketoconazole, selective inhibitors of P4503A enzymes, but not by any of several other P450 inhibitors. Antibodies raised against P4503A4 could inhibit most of the oxidation of losartan to EXP3174 in a microsomal sample having high catalytic activity, but antibodies recognizing other P450s had effect. The oxidation of losartan to EXP3174 was catalyzed by purified human liver microsomal P4504A4 and by purified bacterial recombinant P4503A4. These results provide evidence that P4503A4 (and possibly other P4503A enzymes) play a major role in the formation of an active metabolite EXP3174.

Effects of <i>N</i>²-Alkylguanine, <i>O</i>⁶-Alkylguanine, and Abasic Lesions on DNA Binding and Bypass Synthesis by the Euryarchaeal B-Family DNA Polymerase Vent (exo^–)

Lim, Seonhee,Song, Insil,Guengerich, F. Peter,Choi, Jeong-Yun American Chemical Society 2012 Chemical research in toxicology Vol.25 No.8

<P>Archaeal and eukaryotic B-family DNA polymerases (pols) mainly replicate chromosomal DNA but stall at lesions, which are often bypassed with Y-family pols. In this study, a B-family pol Vent (exo<SUP>–</SUP>) from the euryarchaeon <I>Thermococcus litoralis</I> was studied with three types of DNA lesions<I>N</I><SUP>2</SUP>-alkylG, <I>O</I><SUP>6</SUP>-alkylG, and an abasic (AP) sitein comparison with a model Y-family pol Dpo4 from <I>Sulfolobus solfataricus</I>, to better understand the effects of various DNA modifications on binding, bypass efficiency, and fidelity of pols. Vent (exo<SUP>–</SUP>) readily bypassed <I>N</I><SUP>2</SUP>-methyl(Me)G and <I>O</I><SUP>6</SUP>-MeG, but was strongly blocked at <I>O</I><SUP>6</SUP>-benzyl(Bz)G and <I>N</I><SUP>2</SUP>-BzG, whereas Dpo4 efficiently bypassed <I>N</I><SUP>2</SUP>-MeG and <I>N</I><SUP>2</SUP>-BzG and partially bypassed <I>O</I><SUP>6</SUP>-MeG and <I>O</I><SUP>6</SUP>-BzG. Vent (exo<SUP>–</SUP>) bypassed an AP site to an extent greater than Dpo4, corresponding with steady-state kinetic data. Vent (exo<SUP>–</SUP>) showed ∼110-, 180-, and 300-fold decreases in catalytic efficiency (<I>k</I><SUB>cat</SUB>/<I>K</I><SUB>m</SUB>) for nucleotide insertion opposite an AP site, <I>N</I><SUP>2</SUP>-MeG, and <I>O</I><SUP>6</SUP>-MeG but ∼1800- and 5000-fold decreases opposite <I>O</I><SUP>6</SUP>-BzG and <I>N</I><SUP>2</SUP>-BzG, respectively, as compared to G, whereas Dpo4 showed little or only ∼13-fold decreases opposite <I>N</I><SUP>2</SUP>-MeG and <I>N</I><SUP>2</SUP>-BzG but ∼260–370-fold decreases opposite <I>O</I><SUP>6</SUP>-MeG, <I>O</I><SUP>6</SUP>-BzG, and the AP site. Vent (exo<SUP>–</SUP>) preferentially misinserted G opposite <I>N</I><SUP>2</SUP>-MeG, T opposite <I>O</I><SUP>6</SUP>-MeG, and A opposite an AP site and <I>N</I><SUP>2</SUP>-BzG, while Dpo4 favored correct C insertion opposite those lesions. Vent (exo<SUP>–</SUP>) and Dpo4 both bound modified DNAs with affinities similar to unmodified DNA. Our results indicate that Vent (exo<SUP>–</SUP>) is as or more efficient as Dpo4 in synthesis opposite <I>O</I><SUP>6</SUP>-MeG and AP lesions, whereas Dpo4 is much or more efficient opposite (only) <I>N</I><SUP>2</SUP>-alkylGs than Vent (exo<SUP>–</SUP>), irrespective of DNA-binding affinity. Our data also suggest that Vent (exo<SUP>–</SUP>) accepts nonbulky DNA lesions (e.g., <I>N</I><SUP>2</SUP>- or <I>O</I><SUP>6</SUP>-MeG and an AP site) as manageable substrates despite causing error-prone synthesis, whereas Dpo4 strongly favors minor-groove <I>N</I><SUP>2</SUP>-alkylG lesions over major-groove or noninstructive lesions.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/crtoec/2012/crtoec.2012.25.issue-8/tx300168p/production/images/medium/tx-2012-00168p_0003.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/tx300168p'>ACS Electronic Supporting Info</A></P>