Kinetics of Nucleotide Incorporation Opposite Polycyclic Aromatic Hydrocarbon−DNA Adducts by Processive Bacteriophage T7 DNA Polymerase

A series of six oligonucleotides with dihydrodiol epoxide metabolites of the polycyclic aromatic hydrocarbons (PAHs) benz[a]anthracene and benzo[a]pyrene attached to adenine N6 and guanine N2 atoms were prepared and studied with the processive bacteriophage DNA polymerase T7, exonuclease- (T7-). HIV-1 reverse transcriptase was much less efficient in polymerization than T7-. Benz[a]anthracene and benzo[a]pyrene adducts strongly blocked incorporation of dTTP and dCTP opposite the A and G derivatives, respectively. dATP was preferentially incorporated in all cases. Steady state kinetic analysis indicated that the low catalytic efficiency with adducted DNA was due to both increased Km and lowered kcat values. Some differences due to PAH stereochemistry were observed. Fluorescence estimates of Kd and presteady state kinetic measurements of koff showed no major decrease in the affinity of T7- with damaged DNA substrates or with dNTPs. Presteady state kinetics showed a lack of the normal burst kinetics for dNTP incorporation with all PAH−DNA derivatives. These results indicate that the rate-limiting step is at or before the step of phosphodiester bond formation; release of the oligonucleotide is no longer the slowest step. Thio elemental effects (substitution of α-oxygen with sulfur) were relatively small, in contrast to previous work with T7- and 8-oxo-7,8-dihydroguanine. The effect of these bulky PAH adducts is either to attenuate rates of conformational changes or to introduce an additional conformation problem but not to alter the inherent affinity of the polymerase for DNA or dNTPs

Thiol-Independent DNA Alkylation by Leinamycin

Thiol-Independent DNA Alkylation by Leinamyci

Translesion Synthesis Across Polycyclic Aromatic Hydrocarbon Diol Epoxide Adducts of Deoxyadenosine by <i>Sulfolobus solfataricus</i> DNA Polymerase Dpo4

The mechanisms by which derivatives of polycyclic aromatic hydrocarbons (PAHs) cause mutations have been of considerable interest. Three different N6-adenyl PAH-diol epoxide oligonucleotide derivatives were studied with the archebacterial translesion DNA polymerase Sulfolobus solfataricus Dpo4. Steady-state kinetic analysis indicated insertion of all four dNTPs opposite each of the three N6-adenyl PAH adducts, with only slightly varying misincorporation efficiencies. Full-length extension of shorter primers paired with templates containing the N6-adenyl PAH derivatives proceeded to apparent completion at 45 °C in the presence of added dimethyl sulfoxide. Analysis of the products by high-performance liquid chromatography/collision-induced mass spectrometry indicated the presence of mixtures of products with each PAH adduct. These mixtures correspond to both error-free synthesis and mixtures of polymerization/realignment steps. With an unmodified template, only the expected A:T and G:C pairing was detected in the primer extension products under these conditions, with no frameshifts. These results demonstrate the complexity of polymerization opposite these bulky N6-adenyl PAH adducts, even with a single polymerase

Translesion Synthesis Past the C8- and <i>N</i>²-Deoxyguanosine Adducts of the Dietary Mutagen 2-Amino-3-methylimidazo[4,5-<i>f</i>]quinoline in the <i>Nar</i>I Recognition Sequence by Prokaryotic DNA Polymerases

2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) is found in cooked meats and forms DNA adducts at the C8- and N2-positions of dGuo after appropriate activation. IQ is a potent inducer of frameshift mutations in bacteria and is carcinogenic in laboratory animals. We have incorporated both IQ-adducts into the G1- and G3-positions of the NarI recognition sequence (5‘-G1G2CG3CC-3‘), which is a hotspot for arylamine modification. The in vitro replication of the oligonucleotides was examined with Escherichia coli pol I Klenow fragment exo-, E. coli pol II exo-, and Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4), and the extension products were sequenced by tandem mass spectrometry. Replication of the C8-adduct at the G3-position resulted in two-base deletions with all three polymerases, whereas error-free bypass and extension was observed at the G1-position. The N2-adduct was bypassed and extended by all three polymerases when positioned at the G1-position, and the error-free product was observed. The N2-adduct at the G3-position was more blocking and was bypassed and extended only by Dpo4 to produce an error-free product. These results indicate that the replication of the IQ-adducts of dGuo is strongly influenced by the local sequence and the regioisomer of the adduct. These results also suggest a possible role for pol II and IV in the error-prone bypass of the C8-IQ-adduct leading to frameshift mutations in reiterated sequences, whereas noniterated sequences result in error-free bypass

Noncovalent DNA Binding Drives DNA Alkylation by Leinamycin: Evidence That the <i>Z</i>,<i>E</i>-5-(Thiazol-4-yl)-penta-2,4-dienone Moiety of the Natural Product Serves as an Atypical DNA Intercalator

Molecular recognition and chemical modification of DNA are important in medicinal chemistry, toxicology, and biotechnology. Historically, natural products have revealed many interesting and unexpected mechanisms for noncovalent DNA binding and covalent DNA modification. The studies reported here characterize the molecular mechanisms underlying the efficient alkylation of duplex DNA by the Streptomyces-derived natural product leinamycin. Previous studies suggested that alkylation of duplex DNA by activated leinamycin (<b>2</b>) is driven by noncovalent association of the natural product with the double helix. This is striking because leinamycin does not contain a classical noncovalent DNA-binding motif, such as an intercalating unit, a groove binder, or a polycation. The experiments described here provide evidence that leinamycin is an atypical DNA-intercalating agent. A competition binding assay involving daunomycin-mediated inhibition of DNA alkylation by leinamycin provided evidence that activated leinamycin binds to duplex DNA with an apparent binding constant of approximately 4.3 ± 0.4 × 10³ M^–1. Activated leinamycin caused duplex unwinding and hydrodynamic changes in DNA-containing solutions that are indicative of DNA intercalation. Characterization of the reaction of activated leinamycin with palindromic duplexes containing 5′-CG and 5′-GC target sites, bulge-containing duplexes, and 5-methylcytosine-containing duplexes provided evidence regarding the orientation of leinamycin with respect to target guanine residues. The data allow construction of a model for the leinamycin–DNA complex suggesting how a modest DNA-binding constant combines with proper positioning of the natural product to drive efficient alkylation of guanine residues in the major groove of duplex DNA

Calcium Is a Cofactor of Polymerization but Inhibits Pyrophosphorolysis by the <i>Sulfolobus solfataricus</i> 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. Mg2+ 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 Ca2+, the consequences of replacing Mg2+ with Ca2+ for Dpo4 activity have not been investigated to date. We show here that Ca2+ (but not Ba2+, Co2+, Cu2+, Ni2+, or Zn2+) 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 Mg2+ or Ca2+. Conversely, no pyrophosphorolysis occurs in the presence of Ca2+, although the positions of the two catalytic metal ions at the active site appear to be very similar in mixed Mg2+/Ca2+- and Ca2+-form Dpo4 crystals

Thiol-Activated DNA Damage by α-Bromo-2-cyclopentenone

Some biologically active chemicals are relatively stable in the extracellular environment but, upon entering the cell, undergo biotransformation into reactive intermediates that covalently modify DNA. The diverse chemical reactions involved in the bioactivation of DNA-damaging agents are both fundamentally interesting and of practical importance in medicinal chemistry and toxicology. The work described here examines the bioactivation of α-haloacrolyl-containing molecules. The α-haloacrolyl moiety is found in a variety of cytotoxic natural products including clionastatin B, bromovulone III, discorahabdins A, B, and C, and trichodenone C, in mutagens such as 2-bromoacrolein and 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), and in the anticancer drug candidates brostallicin and PNU-151807. Using α-bromo-2-cyclopentenone (1) as a model compound, the activation of α-haloacrolyl-containing molecules by biological thiols was explored. The results indicate that both low molecular weight and peptide thiols readily undergo conjugate addition to 1. The resulting products are consistent with a mechanism in which initial addition of thiols to 1 is followed by intramolecular displacement of bromide to yield a DNA-alkylating episulfonium ion intermediate. The reaction of thiol-activated 1 with DNA produces labile lesions at deoxyguanosine residues. The sequence specificity and salt dependence of this process is consistent with involvement of an episulfonium ion intermediate. The alkylated guanine residue resulting from the thiol-triggered reaction of 1 with duplex DNA was characterized using mass spectrometry. The results provide new insight regarding the mechanisms by which thiols can bioactivate small molecules and offer a more complete understanding of the molecular mechanisms underlying the biological activity of cytotoxic, mutagenic, and medicinal compounds containing the α-haloacrolyl group

Translesion Synthesis Across 1,<i>N</i>²-Ethenoguanine by Human DNA Polymerases

1,N2-Etheno(ε)guanine (ε) 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,N2-ε-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,N2-ε-G. In this study, we examined the abilities of recombinant, full-length human pol δ and three human translesion DNA pols to copy past 1,N2-ε-G. The replicative pol, pol δ, was completely blocked. Pols ι and κ showed similar rates of incorporation of dTTP and dCTP. Pol η was clearly the most active of these pols in copying past 1,N2-ε-G, incorporating in the order dGTP > dATP > dCTP, regardless of whether the base 5‘ of 1,N2-ε-G in the template was C or T. Pol η also had the highest error frequency opposite 1,N2-ε-G. Analysis of the extended products of the pol η reactions by mass spectrometry indicated only two products, both of which had G incorporated opposite 1,N2-ε-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 η, which appeared to insert G opposite 1,N2-ε-G and then copy correctly. This pattern differs with the same oligonucleotide sequences and 1,N2-ε-G observed using Dpo4, emphasizing the importance of pols in mutagenesis events

Site Specific Synthesis and Polymerase Bypass of Oligonucleotides Containing a 6-Hydroxy-3,5,6,7-tetrahydro-9<i>H</i>-imidazo[1,2-<i>a</i>]purin-9-one Base, an Intermediate in the Formation of 1,<i>N</i>²-Etheno-2‘-deoxyguanosine

The reaction of DNA with certain bis-electrophiles such as chlorooxirane and chloroacetaldehyde produces etheno adducts. These lesions are highly miscoding, and some of the chemical agents that produce them have been shown to be carcinogenic in laboratory animals and in humans. An intermediate in the formation of 1,N2-ethenoguanine is 6-hydroxy-3,5,6,7-tetrahydro-9H-imidazo[1,2-a]purin-9-one (6-hydroxyethanoguanine), which undergoes conversion to the etheno adduct. The chemical properties and miscoding potential of the hydroxyethano adduct have not been previously studied. A synthesis of the hydroxyethano-adducted nucleoside was developed, and it was site specifically incorporated into oligonucleotides. This adduct had a half-life of between 24 and 48 h at neutral pH and 25 °C at the nucleoside and oligonucleotide levels. The miscoding potential of the hydroxyethano adduct was examined by primer extension reactions with the DNA polymerases Dpo4 and pol T7-, and the results were compared to the corresponding etheno-adducted oligonucleotide. Dpo4 preferentially incorporated dATP opposite the hydroxyethano adduct and dGTP opposite the etheno adduct; pol T7- preferentially incorporated dATP opposite the etheno adduct while dGTP and dATP were incorporated opposite the hydroxyethano adduct with nearly equal catalytic efficiencies. Collectively, these results indicate that the hydroxyethano adduct has a sufficient lifetime and miscoding properties to contribute to the mutagenic spectrum of chlorooxirane and related genotoxic species

Distribution of factors significantly associated with LSM failure.

<p>Patients’ percentage distribution according to the factors associated with LSM failure is showed (A), and the significances were determined by univariate and multivariate analyses (B). Intercostal space (IS).</p