Preferential Misincorporation of Purine Nucleotides by Human DNA Polymerase η Opposite Benzo[a]pyrene 7,8-Diol 9,10-Epoxide Deoxyguanosine Adducts

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We have determined the crystal structure of the human base excision repair enzyme DNA polymerase ␤ (Pol ␤) in complex with a 1-nt gapped DNA substrate containing a template N 2 -guanine adduct of the tumorigenic (؊)-benzo[c]phenanthrene 4R,3S-diol 2S,1R-epoxide in the gap. Nucleotide insertion opposite this adduct favors incorrect purine nucleotides over the correct dCMP and hence can be mutagenic. The structure reveals that the phenanthrene ring system is stacked with the base pair immediately 3 to the modified guanine, thereby occluding the normal binding site for the correct incoming nucleoside triphosphate. The modified guanine base is displaced downstream and prevents the polymerase from achieving the catalytically competent closed conformation. The incoming nucleotide binding pocket is distorted, and the adducted deoxyguanosine is in a syn conformation, exposing its Hoogsteen edge, which can hydrogen-bond with dATP or dGTP. In a reconstituted base excision repair system, repair of a deaminated cytosine (i.e., uracil) opposite the adducted guanine was dramatically decreased at the Pol ␤ insertion step, but not blocked. The efficiency of gap-filling dCMP insertion opposite the adduct was diminished by >6 orders of magnitude compared with an unadducted templating guanine. In contrast, significant misinsertion of purine nucleotides (but not dTMP) opposite the adducted guanine was observed. Pol ␤ also misinserts a purine nucleotide opposite the adduct with ungapped DNA and exhibits limited bypass DNA synthesis. These results indicate that Pol ␤-dependent base excision repair of uracil opposite, or replication through, this bulky DNA adduct can be mutagenic.DNA adduct ͉ DNA repair ͉ fidelity ͉ mutagenesis P olycyclic aromatic hydrocarbons (PAHs) such as benzo- , and the adduct derived from trans-opening of the epoxide ring by the exocyclic 2-amino group of guanine in DNA, which is the subject of the present investigation.Bulky PAH-derived lesions can persist in human tissues when cellular repair enzymes fail to correct these lesions (13). The presence of these lesions in the genome blocks replicative DNA polymerases. If these lesions are not repaired, they must be bypassed for replication to continue. Bypass DNA synthesis occurs when a translesional DNA polymerase is recruited to the site of DNA damage and inserts a nucleotide opposite the adduct. DNA synthesis can be error-free or error-prone. The resulting base pair is then extended by either the same DNA polymerase or an auxiliary polymerase. Because replication of these adducts is known to result in mutations (14-16), mutagenic bypass provides a mechanism for adduct-induced tumor initiation and progression.Several DNA repair mechanisms protect cells from the deleterious effects of DNA lesions. For example, bulky DNA adducts such as PAH adducts and the formamidopyrimidine adduct of aflatoxin are primarily repaired by nucleotide excision repair (17, 18), although some unstable B[a]P DE adducts might promote depurination and elicit the base excision repair (BER...

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Structure of DNA polymerase β with a benzo[ c ]phenanthrene diol epoxide-adducted template exhibits mutagenic features

Batra

Shock

Prasad

et al. 2006

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“…30) or interactions with an enzyme's active site may influence the syn-anti conformational preference. Indeed, a possible role for the syn conformation, which can more effectively form hydrogen bonds with an incorrect incoming purine base than with a correct cytosine, has been proposed in erroneous replication of BaP DE dG adducts in DNA (33,34).…”

Section: Discussionmentioning

confidence: 99%

Crystal and molecular structure of a benzo[ a ]pyrene 7,8-diol 9,10-epoxide N ² -deoxyguanosine adduct: Absolute configuration and conformation

Karle

Yagi

Sayer

et al. 2004

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Benzo[a]pyrene 7,8-diol 9,10-epoxide adducts in DNA are implicated in mutagenesis, and their formation from the diol epoxides and subsequent incorrect replication by human DNA polymerases provide an attractive mechanism for the induction of cancer by this highly carcinogenic hydrocarbon and its diol epoxide metabolites. Here, we describe the crystal structure of such an adduct at the exocyclic amino group of a purine nucleoside. The present adduct derives from trans opening at C10 of the (؊)-(7S,8R)-diol (9R,10S)-epoxide enantiomer by the exocyclic N 2 -amino group of deoxyguanosine. In the crystal, the pyrene rings of adjacent molecules stack with each other, but the guanine bases do not stack either intermolecularly with each other or intramolecularly with the pyrene. The most notable features of the molecular structure are (i) independent and unambiguous proof of the absolute configuration of the adduct based on the spatial relationship between the known chiral carbon atoms of the deoxyribose and the four asymmetric centers in the hydrocarbon moiety; (ii) visualization of the relative orientations of the pyrene and guanine ring systems as well as the conformation of the partially saturated hydrocarbon ring (comprising carbon atoms 7, 8, 9, and 10), both of which conformational features in the crystal are in good agreement with deductions from NMR and CD measurements in solution; and (iii) the presence in the crystal of a syn glycosidic torsion angle, a conformation that is unusual in B-DNA but that may be involved in error-prone replication of these benzo[a]pyrene 7,8-diol 9,10-epoxide deoxyguanosine adducts by DNA polymerases.T he polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP), first isolated from coal tar and characterized by Cook et al. in 1933 (1), is one of the most prevalent environmental carcinogens to which humans are exposed (2). It is metabolized in mammals (3) by oxidation on the angular benzo-ring (carbon atoms 7, 8, 9, and 10; Fig. 1) to give two enantiomeric pairs of diastereomeric 7,8-diol 9,10-epoxides (DEs), designated as DE-1 (benzylic 7-hydroxyl group and epoxide oxygen cis; also referred to as the syn diastereomer) and DE-2 (benzylic 7-hydroxyl group and epoxide oxygen trans; also referred to as the anti diastereomer). Of these four DE isomers, (ϩ)-(7R,8S,9S,10R)-DE-2 predominates on metabolism of the hydrocarbon in mammals (3) and is also by far the most tumorigenic isomer in animal models (4). Reaction of BaP DEs with nucleic acids in cells or in vitro occurs mainly by trans and to a lesser extent cis ring opening of their epoxide rings at C10 by the exocyclic 2-and 6-amino groups of the guanine and adenine bases, respectively, to give 16 possible covalent adducts (5-8), with the trans-(10S) deoxyguanosine (dG) adduct derived from the highly tumorigenic to each other, the preferred conformation of the glycosidic bond between the sugar and the purine base, and the conformation of the partially saturated tetrahydro benzo-ring can potentially influence the fit of these adducts in DNA...

“…Recent studies suggest that one or more Y-family DNA polymerases may facilitate either accurate or mutagenic bypass of BPDE adducts (8). For example, polymerase (Pol) is accurate in bypassing BPDE-dG adducts (9,10) whereas Pol is error-prone (11,12). Pol can accurately insert dTMP opposite a BPDE-dA adduct, but then requires Pol to extend primers to complete lesion bypass synthesis (13).…”

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confidence: 99%

Crystal structure of a benzo[ a ]pyrene diol epoxide adduct in a ternary complex with a DNA polymerase

Sayer

Plosky

et al. 2004

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175

230

The first occupation-associated cancers to be recognized were the sooty warts (cancers of the scrotum) suffered by chimney sweeps in 18th century England. In the 19th century, high incidences of skin cancers were noted among fuel industry workers. By the early 20th century, malignant skin tumors were produced in laboratory animals by repeatedly painting them with coal tar. The culprit in coal tar that induces cancer was finally isolated in 1933 and determined to be benzo[a]pyrene (BP), a polycyclic aromatic hydrocarbon. A residue of fuel and tobacco combustion and frequently ingested by humans, BP is metabolized in mammals to benzo[a]pyrene diol epoxide (BPDE), which forms covalent DNA adducts and induces tumor growth. In the 70 yr since its isolation, BP has been the most studied carcinogen. Yet, there has been no crystal structure of a BPDE DNA adduct. We report here the crystal structure of a BPDE-adenine adduct base-paired with thymine at a templateprimer junction and complexed with the lesion-bypass DNA polymerase Dpo4 and an incoming nucleotide. Two conformations of the BPDE, one intercalated between base pairs and another solvent-exposed in the major groove, are observed. The latter conformation, which can be stabilized by organic solvents that reduce the dielectric constant, seems more favorable for DNA replication by Dpo4. These structures also suggest a mechanism by which mutations are generated during replication of DNA containing BPDE adducts.