Deoxyguanosine Residues In DNA Research Articles

Translesion DNA Synthesis by Human DNA Polymerase η on Templates Containing a Pyrimidopurinone Deoxyguanosine Adduct, 3-(2′-Deoxy-β-d-erythro-pentofuranosyl)pyrimido-[1,2-a]purin-10(3H)-one

M1dG (3-(2′-deoxy-β-d-erythro-pentofuranosyl)pyrimido[1,2-a]purin-10(3H)-one) lesions are mutagenic in bacterial and mammalian cells, leading to base substitutions (mostly M1dG to dT and M1dG to dA) and frameshift mutations. M1dG is produced endogenously through the reaction of peroxidation products, base propenal or malondialdehyde, with deoxyguanosine residues in DNA. The mutagenicity of M1dG in Escherichia coli is dependent on the SOS response, specifically the umuC and umuD gene products, suggesting that mutagenic lesion bypass occurs by the action of translesion DNA polymerases, like DNA polymerase V. Bypass of DNA lesions by translesion DNA polymerases is conserved in bacteria, yeast, and mammalian cells. The ability of recombinant human DNA polymerase η to synthesize DNA across from M1dG was studied. M1dG partially blocked DNA synthesis by polymerase η. Using steady-state kinetics, we found that insertion of dCTP was the least favored insertion product opposite the M1dG lesion (800-fold less efficient than opposite dG). Extension from M1dG·dC was equally as efficient as from control primer-templates (dG·dC). dATP insertion opposite M1dG was the most favored insertion product (8-fold less efficient than opposite dG), but extension from M1dG·dA was 20-fold less efficient than dG·dC. The sequences of full-length human DNA polymerase η bypass products of M1dG were determined by LC-ESI/MS/MS. Bypass products contained incorporation of dA (52%) or dC (16%) opposite M1dG or −1 frameshifts at the lesion site (31%). Human DNA polymerase η bypass may lead to M1dG to dT and frameshift but likely not M1dG to dA mutations during DNA replication.

Hepatic iron overload and hepatocellular carcinoma

The liver is the main storage site for iron in the body. Excess accumulation of iron in the liver has been well-documented in two human diseases, hereditary hemochromatosis and dietary iron overload in the African. Hepatic iron overload in these conditions often results in fibrosis and cirrhosis and may be complicated by the development of hepatocellular carcinoma. Malignant transformation usually occurs in the presence of cirrhosis, suggesting that free iron-induced chronic necroinflammatory hepatic disease plays a role in the hepatocarcinogenesis. However, the supervention of hepatocellular carcinoma in the absence of cirrhosis raises the possibility that ionic iron may also be directly hepatocarcinogenic. Support for this possibility is provided by a recently described animal model of dietary iron overload in which iron-free preneoplastic nodules and hepatocellular carcinoma developed in the absence of fibrosis or cirrhosis. The mechanisms by which iron induces malignant transformation have yet to be fully characterized but the most important appears to be the generation of oxidative stress. Free iron generates reactive oxygen intermediates that disrupt the redox balance of the cells and cause chronic oxidative stress. Oxidative stress leads to lipid peroxidation of unsaturated fatty acids in membranes of cells and organelles. Cytotoxic by-products of lipid peroxidation, such as malondialdehyde and 4-hydroxy-2′-nonenal, are produced and these impair cellular function and protein synthesis and damage DNA. Deoxyguanosine residues in DNA are also hydroxylated by reactive oxygen intermediates to form 8-hydroxy-2′-deoxyguanosine, a major promutagenic adduct that causes G:C to T:A transversions and DNA unwinding and strand breaks. Free iron also induces immunologic abnormalities that may decrease immune surveillance for malignant transformation.

Analysis of HPRT and supF mutations caused by pierisin-1, a guanine specific ADP-ribosylating toxin derived from the cabbage butterfly.

Pierisin-1, an ADP-ribosylating toxin derived from the cabbage butterfly, Pieris rapae, induces apoptosis in various mammalian cell lines. We recently reported that the target for ADP ribosylation by pierisin-1 is the 2'-deoxyguanosine residue in DNA. To examine whether pierisin-1 would induce mutations in mammalian cell genes, we conducted a mutational analysis for the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus in pierisin-1-treated Chinese hamster lung (CHL) cells. N(2)-(ADP-ribos-1-yl)-2'-deoxyguanosine was detected by the (32)P-postlabeling method in CHL cells after treatment with pierisin-1 at doses of 2-32 ng/mL; adduct levels were 1.1-12.0 per 10(6) nucleotides. Pierisin-1 induced mutations in the HPRT gene dose-dependently, and the frequency was 38 times higher than the control, at a dose of 32 ng/mL. To confirm that mono(ADP-ribosyl)ated dG itself leads to mutations, the pierisin-1-treated DNA of plasmid pMY189 bearing the supF gene was used for mutational analysis. The mutation frequency of the supF gene treated with 2-8 micro g/mL of pierisin-1 was 17-40-fold the control value. Mutation spectrum analysis showed that single base substitutions dominated in both HPRT and supF genes. Among these, transversions were predominant, and more than 70% of the base substitutions occurred at G:C base pairs in both genes. The most frequent mutations were G:C to C:G, followed by G:C to T:A in HPRT gene, whereas G:C to T:A transversions dominated in the supF gene. Our results indicate that pierisin-1 produced N(2)-(ADP-ribos-1-yl)-2'-deoxyguanosine and this guanine-adduct could lead to mutations in the HPRT and supF genes. These findings could provide very useful information for understanding the biological significance of pierisin-1.

Mono(ADP-ribosyl)ation of 2'-deoxyguanosine residue in DNA by an apoptosis-inducing protein, pierisin-1, from cabbage butterfly.

Pierisin-1 is a potent apoptosis-inducing protein derived from the cabbage butterfly, Pieris rapae. It has been shown that pierisin-1 has an A small middle dotB structure-function organization like cholera or diphtheria toxin, where the "A" domain (N-terminal) exhibits ADP-ribosyltransferase activity. The present studies were designed to identify the target molecule for ADP-ribosylation by pierisin-1 in the presence of beta-[adenylate-(32)P]NAD, and we found DNA as the acceptor, but not protein as is the case with other bacteria-derived ADP-ribosylating toxins. ADP-ribosylation of tRNAs from yeast was also catalyzed by pierisin-1, but the efficiency was around 110 of that for calf thymus DNA. Pierisin-1 efficiently catalyzed the ADP-ribosylation of double-stranded DNA containing dG small middle dotdC, but not dA small middle dotdT pairs. The ADP-ribose moiety of NAD was transferred to the amino group at N(2) of 2'-deoxyguanosine to yield N(2)-(alpha-ADP-ribos-1-yl)-2'-deoxyguanosine and its beta form, which were determined by several spectral analyses including (1)H- and (13)C-NMR and mass spectrometry. The chemical structures were also ascertained by the independent synthesis of N(2)-(D-ribos-1-yl)-2'-deoxyguanosine, which is the characteristic moiety of ADP-ribosylated dG. Using the (32)P-postlabeling method, ADP-ribosylated dG could be detected in DNA from pierisin-1-treated HeLa cells, in which apoptosis was easily induced. Thus, the targets for ADP-ribosylation by pierisin-1 were concluded to be 2'-deoxyguanosine residues in DNA. This finding may open a new field regarding the biological significance of ADP-ribosylation.

Impact of Site-Specific Benzo[a]Pyrene Diol Epoxide-dG Lesions at or near Single/Double-Strand DNA Junctions on DNA Bending

Adducts derived from the binding of the (+)-7R,8S,9S,10R and (−)-7S,8R,9R,10S enantiomers of r7,t8-dihydrodiol-t9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) to 2′-deoxyguanosine residues in DNA are known to induce mutations due to error-prone DNA replication. Because the conformational properties of these lesions may be important in these phenomena, we have examined the effects of the stereoisomeric (+)- and (−)-trans-anti-[BP]-N 2-dG lesions positioned site-specifically at or near primer/template oligonucleotide junctions on DNA bending using high resolution gel electrophoresis. Remarkable differences in electrophoretic mobilities μ are observed in the two adducts derived from the tumorigenic (+)-anti-BPDE, and the non-tumorigenic (−)-anti-BPDE enantiomer. With the (+)-trans lesion positioned on the template strand adjacent to the 3′-end of the primer strand, a remarkable decrease in μ is observed. This suggests the existence of a bend at the single strand-double strand junction. Only modest decreases in μ are observed in the case of the (−)-trans lesion, or when the 3′-end is opposite to, or more distant from the lesion site. These observations are discussed in terms of the known NMR solution structures of these lesions in the same sequence context, and the properties of primer/template DNA in polymerases.

Quantitative reactions of anti 5,9-dimethylchrysene dihydrodiol epoxide with DNA and deoxyribonucleotides

Native as well as denatured calf thymus DNA, deoxyguanylic and deoxyadenylic acid, respectively, were reacted with the racemic anti 5,9-dimethylchrysene dihydrodiol epoxide (5,9-DMCDE). The deoxyribonucleoside adducts were separated by HPLC and characterized by CD and NMR. Approximately 17% of the epoxide was trapped by native DNA and 76% of the adducts were derived from the RSSR enantiomer. The ratios of dAdo/dGuo modification in DNA were 14/86 and 19/81 for RSSR and SRRS enantiomers, respectively. By monitoring the product yields of anti 5,9-DMCDE with DNA and deoxyribonucleotides, we hoped to gain further insight into the factors responsible for deoxyguanosine adduct formation by 5-methylchrysene dihydrodiol epoxide (5-MCDE) compared to 5,6-dimethylchrysene dihydrodiol epoxide (5,6-DMCDE). The adduct yields in deoxyribonucleotide reactions of 5,9-DMCDE were slightly higher than those from 5-MCDE. However, the reaction yields of 5,9-DMCDE with DNA were lower than those with 5-MCDE in most cases, particularly for the cis and trans deoxyadenosine adducts. It seems that the 9-methyl group of 5,9-DMCDE significantly influences adduct formation with the deoxyadenosine residue in DNA in contrast to the 6-methyl group of 5,6-DMCDE. The 9-methyl group sterically decreases deoxyadenosine adduct yields more in reaction with native DNA than denatured DNA, but it has little effect on deoxyribonucleotide reactions. Adduct formation with deoxyguanosine residues in DNA by all three dihydrodiol epoxides correlate with their respective tumorigenic and mutagenic activities.

Pyrimido[1,2-alpha]purin-10(3H)-one: a reactive electrophile in the genome.

Malondialdehyde and base propenal react with deoxyguanosine residues in DNA to form an exocyclic adduct, pyrimido[1, 2-alpha]purin-10(3H)-one (1), that has been detected at high levels in genomic DNA of healthy humans. Previous studies have shown that tris(hydroxymethyl)aminomethane adds to 1 at elevated pH, forming an enaminoimine (2), but it is uncertain whether 1 reacts directly or hydrolyzes under basic conditions to N(2)-(3-oxo-1-propenyl)deoxyguanosine (3) prior to amine addition. We report that 1 reacts at neutral pH with hydroxylamines to form oximes. The rate of reaction of 1 with hydroxylamines at pH 7 is at least 150 times faster than the rate of hydrolysis of 1 to 3. Thus, 1 is directly reactive to nucleophiles. These observations indicate that 1 is an electrophile in the human genome that may react with cellular nucleophiles to form novel cross-linked adducts.

7,8-Dihydro-8-oxo-2‘-deoxyguanosine Residues in DNA Are Radiation Damage “Hot” Spots in the Direct γ Radiation Damage Pathway

7,8-Dihydro-8-oxo-2‘-deoxyguanosine Residues in DNA Are Radiation Damage “Hot” Spots in the Direct γ Radiation Damage Pathway

Mutational specificity of the syn 1,2-dihydrodiol 3,4-epoxide of 5-methylchrysene.

The mutational specificity of the syn dihydrodiol epoxide of 5-methylchrysene in the supF gene of the pSP189 vector was examined. Transversion mutations at GC pairs predominated with G --> T and G --> C changes accounting for 42 and 21% of total base change mutations. The types of mutations found reflect the previously determined chemical preference of this reactive species for reaction with deoxyguanosine residues in DNA.

NMR studies of a DNA containing 8-hydroxydeoxyguanosine

The effects of hydroxylation at the C8 of a deoxyguanosine residue in DNA were studied by NMR analysis of a self-complementary dodecanucleotide, d(C1-G2-C3-oh8G4-A5-A6-T7-T8-C9-G10-C11-G12), which has an 8-hydroxy-2'-deoxyguanosine (oh8dG) residue at the 4th position. NMR data indicate that the 8-hydroxyguanine (oh8G) base takes a 6,8-diketo tautomeric form and is base-paired to C with Watson-Crick type hydrogen bonds in a B-form structure. The thermal stability of the duplex is reduced, but the overall structure is much the same as that of the unmodified d(CGCGAATTCGCG) duplex. The structural changes caused by 8-hydroxylation of the deoxyguanosine, if any, are localized near the modification site.

Misreading of DNA templates containing 8-hydroxydeoxyguanosine at the modified base and at adjacent residues.

It has been shown previously that deoxyguanosine residues in DNA are hydroxylated at the C-8 position both in vitro and in vivo to produce 8-hydroxydeoxyguanosine (8-OH-dG) by various agents that produce oxygen radicals such as reducing reagents-O2, metal ions-O2, polyphenol-H2O2-Fe3+, asbestos-H2O2 or ionizing radiation. These agents are mostly either mutagenic or carcinogenic; therefore, the formation of 8-OH-dG can also be considered a likely cause of mutation or carcinogenesis by oxygen radicals. It is of interest to know whether the 8-OH-dG residue in DNA is misread during DNA replication. To answer this question, we have examined the effect of the 8-OH-dG residue in DNA on the fidelity of DNA replication using a DNA synthesis system in vitro with Escherichia coli DNA polymerase I (Klenow fragment). The synthetic oligodeoxynucleotides, with or without an 8-OH-dG residue in a specified position, were chemically synthesized and used as templates for DNA synthesis under the conditions of the dideoxy chain termination sequencing method. Surprisingly, in addition to misreading of the 8-OH-dG residue itself, pyrimidines next to the 8-OH-dG residue (G has not yet been tested) were also misread.

Metabolic activation of benzo[ a]pyrene in human skin maintained in short-term organ culture

The metabolic activation of benzo[ a]pyrene (BP) was examined in six samples of human skin after topical application of the hydrocarbon to the skin in short-term organ culture. The results show that all of the samples were capable of metabolizing BP to water-soluble products and to ethersoluble products that included the 4,5-, 7,8- and 9,10-dihydrodiols and a product which had chromatographic properties identical with those of authentic trans-11,12-dihydro-11,12-dihydroxybenzo[ a]pyrene (BP-11,12-diol). The major BP-deoxyribonucleoside adduct detected in each skin sample appeared to be formed from the reaction of r-7, t-8-dihydroxy- t-9,10-oxy-7,8,9,10-tetrahydrobenzo[ a]pyrene ( anti-BP-7,8-diol 9,10-oxide) with deoxyguanosine residues in DNA.

Differentiation of DNA-platinum complexes by fluorescence. The use of an intercalating dye as a probe.

Ethidium bromide (EtdBr) was used to differentiate perturbations induced in salmon sperm DNA complexed with a series of platinum compounds. The DNA · Pt complexes were classified into two groups. First, those which show a DNA · Pt · EtdBr fluorescence almost identical to that of the DNA · EtdBr complex and second, those which decrease linearly the fluorescence of the EtdBr molecules upon platinum fixation. In the first group, the platinum compounds are fixed to DNA at only one site, like [Pt(dien)Cl]Cl and [Pt(NH3)3Cl]Cl. In this case, the platinum fixation corresponding to rb= 0.20 induced a 15–20% fluorescence decrease (rb= number of Pt atoms bound per nucleotide). On the other hand cis-Pt(NH3)2Cl2, cis-[Pt(NH3)2(H2O)2] (NO3)2, cis-Pt(en)Cl2, cis-[Pt(en)(H2O)2](NO3)2, K[Pt(NH3)-Cl3] and K2[PtCl4] were found to be of the second group. These platinum compounds react with DNA through chelation; the fluorescence decrease was found to be 70% for rb= 0.20. trans-Pt-(NH3)2Cl2 can fit into either the first group, when one chlorine atom is still covalently fixed on platinum (one DNA · Pt bond), or into the second one, when the chlorine atoms are displaced (trans-bidentate fixation). Scatchard plots indicated that when one Pt compound is fixed to DNA by chelation it inhibited the intercalation of one EtdBr molecule, this relationship being verified for rb≤ 0.10. The binding constant of EtdBr to the different DNA · Pt complexes was not altered. Since no fluorescence quenching was detected in the DNA · Pt · EtdBr complexes, the fluorescence decrease was attributed to the so-called ‘chelation effect’ and interpreted in terms of local denaturations of the DNA molecule. The perturbations could be due to the rupture of the hydrogen bonds in dG · dC pairs since chelation should modify the deoxyguanosine residues in DNA.

Mechanism of cellular drug resistance.

CELLULAR resistance to drugs or chemicals, which can be “natural”1–4 or “acquired”5,6, is important particularly in the treatment of infectious diseases by antibiotics and of tumour cells by cancer chemotherapeutic drugs. I have investigated the mechanism of cellular drug resistance using mammalian cell lines resistant to actinomycin D. In murine leukaemias7, HeLa cells8 and bacteria9 this resistance has been related to membrane permeability barriers which prevent the interaction of actinomycin D with deoxyguanosine residues in DNA, stopping its pharmacological action. Earlier I had found that L5178Y (mouse lymphoma cell line) and L5178Y/D (actinomycin D-resistant subline) glycosidase10 and glycoprotein: glycosyl transferase11 activities, which are important in membrane glycoprotein catabolism and anabolism, were altered in the resistant lines. I have now shown that surface membranes are affected, particularly the glycoproteins and glycolipids and their synthesis in the cells resistant to actinomycin D. In this article I shall develop the hypothesis that acquired drug resistance occurs through selection of cells with altered membranes and altered membrane synthetic apparatus, and that such membrane changes decrease drug permeability and thus increase drug resistance.