O6 Of Guanine Research Articles

Sequence specificity of guanine alkylation and repair.

The sequence selectivity of methylation at the O6 and N7 position of guanine by N-methyl-N'-nitrosourea (MNU) and the rate of removal of O6-methylguanine by O6alkylguanine-DNA alkyltransferase (AGT) was determined using dodecadeoxynucleotides of defined structure. The extent of guanine adduct formed in self-complementary dodecamers, 5'-TATACGCGTATA-3', 5'-TATACCGGTATA-3' and 5'-TATAGGCCTATA-3', after methylation with [3H]MNU in a representative experiment were, respectively, 10, 19 and 30 pmol O6-methylguanine/mumol guanine and 97, 189 and 217 pmol N7-methylguanine/mumol guanine. The O6-methylguanine/N7-methylguanine ratio remained relatively constant for each dodecamer. A direct comparison between the methylation at guanine with adenine or thymine as the 5'-flanking base was made with two dodecamers, 5'-TATACATGTATA-3' and 5'-TATACTAGTATA-3'. When the guanine residue was preceded 5' by an adenine, the level of O6 and N7-alkylation was, respectively, 2.1-fold and 1.5-fold greater than when guanine was preceded 5' by a thymine. These date are consistent with a regioselective mechanism for alkylnitrosourea alkylation of guanine. The methylated dodecamer, 5'-TATACGCGTATA-3' was repaired faster than 5'-TATACCGGTATA-3' by HT29 extract containing AGT with a loss in 10 min of 0.052 pmol and 0.025 pmol O6-methylguanine, respectively. Dodecamers of the structure 5'-dCGCGAATTCm6GCG-3' and 5'-dCGCCAATTGm6GCG-3' were labeled at the 5' end with 32P by the reaction with polynucleotide kinase and after incubation with AGT, the methylated and demethylated dodecamers were separated by reversed-phase HPLC. The amount of demethylated product formed was greater for the dodecamer containing cytosine as the 5'-flanking base to O6-methylguanine compared to guanine in that same position. A higher extent of alkylation by MNU and a slower rate of repair by AGT for sites in which a guanine or modified guanine is preceded by a purine rather than a pyrimidine may explain, at least in part, mutational hot spots.

An MNDO molecular orbital study of the reactions of protonated oxirane with guanine

The bimolecular hydroxyalkylation of the N2, 3-, O6, and 7-position of guanine by protonated oxirane has been studied using the MNDO molecular orbital procedure. The enthalpies of activation (relative to the isolated reactants) were calculated to be 12.5, 11.4, −4.8, and −4.9 kcal mol−1, respectively. The transition state geometries were characteristically SN l-like. The forming bonds reach ca. 0–2% of their final strengths while cleavage of the breaking bonds is ca. 77–87% complete. Their relative energies are dominated by electrostatic interactions between the reacting moieties with little to no charge transfer involved. The relevance of these results to the reactions of carcinogenic oxiranes and their derivatives with nucleic acids is discussed.

Thymine and guanine base specificity of human myeloma proteins with anti-DNA activity.

To further our understanding of the molecular basis of DNA-autoantibody interactions, we have characterized the specificities of three IgG human myeloma proteins that bind DNA. We measured their binding to synthetic single- and double-stranded homopolynucleotides, random and alternating copolymers, oligonucleotides, and nucleotides or nucleosides conjugated to non-nucleic acid carriers. All three antibodies bound single-stranded nucleic acids, including both polyribonucleotides and polydeoxyribonucleotides. They varied in relative affinities for polynucleotides of varying base composition. Polymers containing the purines guanine or hypoxanthine and/or the pyrimidine thymine were most reactive with all three proteins. A myeloma protein that reacted with poly(G), poly(I), or poly(dT) also bound to the corresponding nucleosides or nucleotides conjugated to bovine serum albumin. None of the antibodies reacted with base-paired double-helical polynucleotides (double-stranded RNA, RNA-DNA hybrid or double-stranded DNA). The results indicate that base specificity is prominent in their reactions and that the accessible epitopes in single-stranded polynucleotides become masked upon base pairing in double-stranded helices. These findings suggest a model in which positions N1 and O6 of guanine and hypoxanthine and N3 and O4 of thymine interact with amino acids of the antibody-combining site.

Reaction of DNA with chemically or enzymatically activated mitomycin C: isolation and structure of the major covalent adduct.

The antitumor antibiotic mitomycin C is shown to form a covalent complex with calf thymus DNA under anaerobic conditions in the presence of either NADPH cytochrome c reductase/NADPH, xanthine oxidase/NADH, or the chemical reducing system H2/PtO2. Digestion of the complex with DNase I/snake venom diesterase/alkaline phosphatase yields a single mitomycin deoxyguanosine adduct as the major DNA alkylation product, identified as N2-(2'' beta,7''-diaminomitosen-1'' alpha-yl) 2'-deoxyguanosine (Structure 2). Two minor adducts, 2-5% each of the total adduct pool, are isolated and identified as the 1'' beta stereoisomer of 2 (Structure 3), and 10''-decarbamoyl-2 (Structure 7). The same results were obtained with M13 DNA and poly(dG-dC).poly(dG-dC); however, in the latter case, a minor adduct apparently possessing two deoxyguanosine and one mitomycin unit is isolated. Digestion of the covalent mitomycin-calf thymus DNA complex with nuclease P1 yields four dinucleotide adducts, all of which consist of 2 linked at its 3' end to each of the four possible 5' nucleotides (A, T, G, and C). Upon treatment of each dinucleotide adduct with snake venom diesterase/alkaline phosphatase, 2 is released along with the corresponding free nucleoside. In apparent conflict with the present results, previous reports from another laboratory have indicated that modification of calf thymus DNA by mitomycin C under conditions identical to those described here result in the isolation of three mitomycin C mononucleotide adducts possessing linkages of the drug to N2 and O6 of guanine and N6 of adenine. Evidence is shown suggesting that the latter adducts are actually three of the above four dinucleotide derivatives of 2 obtained independently by us and, thus, all of them in fact possess an identical N2-mitosenylguanine adduct moiety. Model-building studies indicate an excellent fit of the guanine N2-linked drug molecule inside the minor groove of B-DNA with no appreciable distortion of the DNA structure.

DNA ethylations induced by ethylnitrosourea in the wild type, cdc4 and cdc7 strains of Saccharomyces cerevisiae.

The experiments reported here have investigated the induction of ethylations to DNA in yeast cells exposed to the chemical mutagen ethylnitrosourea. A similar level of alkylation was seen at the N7 and O6 of guanine and at the N3 of adenine in either log phase cells or in temperature-sensitive cdc4 and cdc7 cells growth arrested at their specific G1 positions. Hence the changes in chromosome structure associated with the above cdc phenotypes do not modify the amount of DNA damage induced by ethylnitrosourea.

The effect of spermine binding on the reactivity of DNA towards carcinogenic alkylating agents.

The effect of spermine binding on the electrostatic potential of DNA is evaluated. The calculations are performed for the essential reactive sites, atoms N7 and O6 of guanine, N3 and N7 of adenine, of the nucleic acid and for its surface envelope. An important weakening of the potential is found affecting all the important reactive sites in both grooves and spreading moreover along the polynucleotide chain far away from the site of binding of the ligand. These results are discussed in connection with the experimentally observed inhibitory effect of spermine binding on DNA methylation by carcinogenic agents.

The effect of methylation of the 6 oxygen of guanine on the structure and stability of double helical DNA.

The effect of methylation of the O-6 position of guanine in short segments of double helical DNA has been investigated by molecular mechanical simulations on the sequences d(CGCGCG)2, d(CGC[OMG]CG)2, d(CGT[OMG]CG)2, d(CGC[OMC]CG/(CGCGCG), d(CGC[OMG]CG/d(CGTGCG), d(CGCGAATTCGCG)2 and d(CGCGAATTC[OMG]CG)2. Guanines methylated at the O-6 position are found to form hydrogen bonds of roughly equal strength to cytosine and thymine. The optimum structure of these modified base pairs are not dramatically different from normal GC pairs, but both involve some bifurcation of the proton donors of cytosine (4NH2) or thymine (3NH) between the guanine N3 and O6 groups.

Structural basis for stabilization of Z-DNA by cobalt hexaammine and magnesium cations.

In the equilibrium between B-DNA and Z-DNA in poly(dC-dG), the [Co(NH3)6]3+ ion stabilizes the Z form 4 orders of magnitude more effectively than the Mg2+ ion. The structural basis of this difference is revealed in Z-DNA crystal structures of d(CpGpCpGpCpG) stabilized by either Na+/Mg2+ or Na+/Mg2+ plus [Co(NH3)6]3+. The crystals diffract X-rays to high resolution, and the structures were refined at 1.25 A. The [Co(NH3)6]3+ ion forms five hydrogen bonds onto the surface of Z-DNA, bonding to a guanine O6 and N7 as well as to a phosphate group in the ZII conformation. The Mg2+ ion binds through its hydration shell with up to three hydrogen bonds to guanine N7 and O6. Higher charge, specific fitting of more hydrogen bonds, and a more stable complex all contribute to the great effectiveness of [Co(NH3)6]3+ in stabilizing Z-DNA.

Specificity in carcinogen-DNA interaction: A theoretical exploration of the factors involved in the effect of neighboring bases on N-methyl- N-nitrosourea alkylation of DNA

Recent experimental studies indicate that in a polynucleotide chain neighboring bases have a significant effect on the relative alkylation of O6 or N7 of guanine by N-methyl-N-nitrosourea (MNU). This paper provides a theoretical exploration of this phenomenon in terms of an appropriate index of reactivity, called accessible surface integrated field (ASIF), introduced recently for the very sake of accounting for specificity or selectivity in drug-macromolecule interaction. The detailed analysis indicates that in the present case the observed variations in relative reactivity are attributable essentially to parallel variations in the accessibilities to the target atoms.

Crystallographic Study of Mechanism of Ribonuclease Ti-Catalysed Specific RNA Hydrolysis

Ribonuclease T1 (RNase T1) cleaves the phosphodiester bond of RNA specifically at the 3'-end of guanosine. 2'-guanosinemonophosphate (2'-GMP) acts as inhibitor for this reaction and was cocrystallized with RNase T1. X-Ray analysis provided insight in the geometry of the active site and in the parts of the enzyme involved in the recognition of guanosine. RNase T1 is globular in shape and consists of a 4.5 turns alpha-helix lying "below" a four-stranded antiparallel beta-sheet containing recognition center as well as active site. The latter is indicated by the position of phosphate and sugar residues of 2'-GMP and shows that Glu58, His92 and Arg77 are active in phosphodiester hydrolysis. Guanine is recognized by a stretch of protein from Tyr42 to Tyr45. Residues involved in recognition are peptide NH and C = O, guanine O6 and N1H which form hydrogen bonds and a stacking interaction of Tyr45 on guanine. Although, on a theoretical basis, many specific amino acid-guanine interactions are possible, none is employed in the RNase T1.guanine recognition.

A theoretical evaluation of the effect of netropsin binding on the reactivity of DNA towards alkylating agents.

The effect of netropsin binding on the electrostatic potential of DNA reactive sites is presented. Calculations are performed for atoms N7 and O6 of guanine, N3 and N7 of adenine of model, 25 base pair long, DNA-netropsin complexes. An important weakening of the potential is found spreading along all the oligonucleotide chain studied. The results are discussed in connection with the inhibitory effect of a related ligand, distamycin A, on DNA methylation.

The influence of small monovalent cations on the hydrogen bonds of base pairs of DNA

The influence of small monovalent metal ions on the multiple hydrogen bonds of Watson-Crick DNA base pairs has been theoretically studied, using ab initio calculations with minimal GLO basis set, and combined with the Boys-Bernardi counterpoise method to eliminate the basis set superposition error. The results were compared with the available experimental data. Only metal ion binding to N3 or N7··O6 of guanine seems to lead to a hydrogen bond stabilization of the GC pair, whereas in the case of AT pair no such effect could be observed.

Alkylation of DNA and tissue specificity in nitrosamine carcinogenesis.

A peculiarity of nitrosamines is the high degree of cell and organ specificity in inducing tumors. There is substantial evidence that the initiation of the carcinogenesis process by carcinogens of this group is linked to the metabolic competence of the target tissue or cell to convert these carcinogens into mutagenic metabolites and to the binding of those metabolites to cellular DNA. Alkylation occurs in the DNA at the N-1, N-3, and N-7 positions of adenine; the N-3, N-7, and O6 of guanine; the N-3, and O2 of cytosine; and the N-3, O4, and O2 of thymine; and the phosphate groups. The initial proportion of each DNA adduct depends upon the alkylating agent used. The various DNA adducts are lost to a variable extent from DNA in vivo by spontaneous release of bases and/or by specific DNA repair processes. Studies conducted in vitro and vivo indicate that alkylation at the oxygen atoms of DNA bases is more critical than alkylation at other positions in the mutagenesis and carcinogenesis induced by N-nitroso compounds. In particular, tissues in which tumors occur more frequently after a pulse dose of nitrosamine are those in which O6-alkylguanine persists longest in DNA, presumably resulting in an increased probability that a miscoding event (mutation) will take place during DNA synthesis. The more rapid removal of O6-methylguanine from the DNA of liver (as compared with extrahepatic tissues) of rats has been associated with the absence of tumor production in this organ by a single dose of dimethylnitrosamine; however, a significant incidence of liver tumors is observed if the same dose is given 24 hr after partial hepatectomy, and tumors are induced by such a dose of dimethylnitrosamine in the liver of hamsters, which has a low capacity to remove O6-methylguanine from its DNA. These data also indicate that the rate of disappearance of 7-methylguanine from the liver or extrahepatic tissues is independent of the dose of dimethylnitrosamine; whereas O6-methylguanine is lost from DNA more rapidly after a low dose of this nitrosamine. It has been shown that in liver the removal of O6-methylguanine but not other DNA adducts, from DNA can be affected by pretreating the animals with N-nitroso compounds. The modulation of DNA repair processes observed after a single dose and after chronic treatment with nitrosamines is discussed in relation to the tissue-specific carcinogenic effect of this group of carcinogens.

Chemical reactivity of a methyldiazonium ion with nucleophilic centers of DNA bases

A series of hierarchical chemical reactivity calculations have been performed to elucidate the alkylation properties of a methyldiazonium ion toward DNA base sites. Both MINDO/3 and CNDO/2 approximate methods have been employed. For the isolated bases the O6 of guanine is predicted to be the most reactive site. This prediction may also be relevant to single-stranded DNA chains containing guanine. For base-pairs, the N7 and O6 sites on guanine are about equally favored for alkylation. The previous study of aziridinium ion alkylation gave about the same results with N7 guanine modestly favored as the preferred site of alkylation for base-pairs. In composite we conclude that N7 guanine and/or O6 guanine will be the preferred sites for alkylation by a methyldiazonium ion but cannot distinguish between these two in terms of chemical specificity.

The relationship between alkylation of specific DNA bases and induction of sister chromatid exchange.

The mechanism of induction of sister chromatid exchange (SCE) was investigated by treating Chinese hamster V-79 cells with two ethylating and two methylating mutagens at doses, taken from linear response curves, that produced 30 SCE/cell. Concentrations of the DNA alkylation products were measured or calculated at 11 DNA base sites and at the phosphodiester bond. Ethyl methanesulfonate, N-methyl- and N-ethyl-N-nitrosourea produced comparable concentrations (3.3 to 3.5 micromol product/mol DNA phosphate) of O6-alkylguanine. Hence, alkylation at O6 of guanine appears relevant to SCE induction for these mutagens. Since alkylation at O6 of guanine has been positively correlated with mutagenesis in V-79 cells, these findings support the suggestion that SCE and mutagenesis can result from a common DNA lesion. Methyl methanesulfonate (MMS) produced very little O6-methylguanine, but did produce 3-methylthymine and 3-methyladenine, either of which might account for the MMS-induced SCE. Thus, for a series of mutagens, induction of SCE does not necessarily result from a single specific DNA lesion. Therefore, SCE can be considered a qualitative indicator of potential mutagenic events.

Interaction with Nucleic Acids of Carcinogenic and Mutagenic N-Nitroso Compounds

Publisher Summary This chapter describes that in the case of mutagenesis, it is likely that alkylation of DNA at certain sites leads to mutations, and at most other sites to variable degrees of toxicity. The important sites of alkylation have not been established, but probably N-7 of guanine is not one of them; O6 of guanine or N-3 of adenine is more likely. It is also possible, because of some recent results, that interaction of the whole molecule, following activation, is an initial reaction of nitrosamines, and possibly even of nitrosamides. Intramolecular rearrangements during replication of the DNA might then lead to fixation of the lesion as a mutation. The relation of the reaction of nitroso compounds with nucleic acids to carcinogenesis is less decisive, and it cannot be said that any good correlations, withstanding rigorous tests, have been established. The existence of a deuterium isotope effect in carcinogenesis by nitrosamines implies that one of the initial steps in carcinogenesis is loss of a hydrogen atom alpha to the nitroso function. This is explainable in terms of formation of a simple alkylating agent from dimethylnitrosamine. However, other types of interaction of the latter with nucleic acids are certainly compatible with the lesser carcinogenicity of the deuterium-labeled compound. Such interactions could damage DNA, and differences in rates of repair that have been found are conceivable as responsible for the differential carcinogenic effects of various nitroso compounds. The chapter describes that correlations must be made with organ specificities and with differences in biological effect in different species. Minor differences in rate of some reaction must be related with sharp, all-ornothing differences in tumor response of one organ versus another.