Bending and circularization of site-specific and stereoisomeric carcinogen-DNA adducts

The potent tumorigen and mutagen (+)-7(R),8(S)-dihydroxy-9(S), 10(R)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene ((+)-anti-BPDE) is a metabolite of benzo[a]pyrene that binds predominantly to the exocyclic amino group of guanine residues in DNA in vivo and in vitro. While the (-)-7S,8R,9R,10Senantiomer, (-)-anti-BPDE, also reacts with DNA to form similar covalent N2-deoxyguanosyl adducts, this diol epoxide is nontumorigenic and its mutagenic activities are different from those of (+)-anti-BPDE. In this work, T4 ligase-induced cyclization methods have been employed to demonstrate that the (+)-anti-[BP]-N2-dG lesions (G*) cause significantly greater amounts of bending and circularization of the one-base overhang undecamer duplex 5'-d(CACAT[G*]TACAC).d(TGTACATGTGG) than the stereoisomeric oligonucleotide duplex with G* = (-)-anti-[BP]-N2-dG. In the case of the (+)-anti-BPDE-modified oligonucleotides, the ratio of circular to linear DNA multimers reaches values of 8-9 for circle contour sizes of 99-121 base pairs, while for the (-)-anti-[BP]-N2-dG-modified DNA this ratio reaches a maximum value of only approximately 1 at 154-176 base pairs. Assuming a planar circle DNA model, the inferred bending angles for 90-92% of the observed circular ligation products range from 30 to 51 degrees per (+)-trans-anti-[BP]-N2-dG lesion and from 20 to 40 degrees per (-)-trans-anti-[BP]-N2-dG lesion. In the case of unmodified DNA, the probability of circular product formation is at least 1 order of magnitude less efficient than in the BPDE-modified sequences and about 90% of the circular products exhibit bending angles in the range of 14 -19 degrees . In the most abundant circular products observed experimentally, the bending angles are 40 degrees and 26 +/- 2 degrees per (+)-anti-[BP]- or (-)-anti-[BP]-modified 11-mer; these values correspond to a net contribution of 21-26 degrees and 5-19 degrees , respectively, to the observed overall bending per lesion. The coexistence of circular DNA molecules of different sizes and, therefore, different average bending angles per lesion, suggest that the lesions induce both torsional flexibility and flexible bends, which permit efficient cyclization, especially in the case of (+)-trans-[BP]-N2-dG adducts. The NMR characteristics of (+)-trans-[BP]-N2-dG lesion in the 11-mer duplex 5'-d(CACAT[G*]TACAC).d(GTGTACATGTG) indicate that all base pairs are intact, except at the underlined base pairs. This suggests a distortion in the normal conformation of the duplex on the 5'-side of the modified guanosine residue, which may be due to bending enhanced base pair opening and bending induced by the bulky carcinogen residue. The implications of base sequence-dependent flexibilities and conformational mobilities of anti-[BP]-N2-dG lesions on DNA replication and mutation are discussed.     

Reactivity of haloketenes and halothioketenes with nucleobases: chemical characterization of reaction products

Halothioketenes and haloketenes are postulated as intermediates in haloolefin bioactivation. Little is known about the interactions of these reactive intermediates with macromolecules such as DNA. DNA binding, however, may be relevant in the toxicity of the parent olefins since they or their proximate metabolites are genotoxic. This prompted us to elucidate the structures and properties of potential DNA adducts formed. Adenine, cytosine, guanine, and thymine were reacted with chloro- and dichlorothioketene, chloro- and dichloroketene, and chloro- and dichloroacyl chloride. While thymine did not react, adenine and cytosine formed stable DNA base adducts with all reaction partners as demonstrated by HPLC analysis. Guanine yielded only products with chloroketene and chloroacetyl chloride. The pH-dependent UV spectra, 1H and 13C NMR, FT-IR, and elemental analysis showed (i) nucleophilic attack of the exocyclic amino groups of the DNA bases yielded haloacyl (thio)amides with all reactants as clearly demonstrated by the FT-IR spectra; (ii) the sulfur in the initial thioamides seems to be rapidly exchanged with oxygen; (iii) the acyl chlorides form identical products but in lower yields as compared to the haloketenes. Reactions of the nucleosides with haloketenes showed the formation of similar nucleoside adducts upon HPLC and MS analysis. Beside the modification of the base moieties, additional peaks in the reaction mixtures analyzed suggested acylation of the deoxyribose hydroxyl groups. In aqueous solutions at pH 7 N6-(chloroacetyl)adenine, N4-(chloroacetyl)cytosine, and N2-(chloroacetyl)guanine are not stable and cleaved to the original base or form 1,N6-acetyladenine, 3,N4-acetylcytosine, 1,N2-acetylguanine, and N2,3-acetylguanine. Under the same conditions, N6-(dichloroacetyl)adenine and N4-(dichloroacetyl)cytosine were completely hydrolyzed to adenine and cytosine, respectively. All haloacyl DNA base adducts proved to be stable at pH 5 but were rapidly degraded at neutral or alkaline pH. The compounds with an additional five-membered ring remained unchanged after 1 week at room temperature. All synthesized DNA base adducts except N2-(chloroacetyl)guanine and 1,N2-acetylguanine were fluorescent. The characterized compounds, especially the etheno (epsilon) base adduct-related derivatives, may represent potential DNA adducts formed as a consequence of haloolefin bioactivation.     

Refined crystal structures of guanine nucleotide complexes of adenylosuccinate synthetase from Escherichia coli

Structures of adenylosuccinate synthetase from Escherichia coli complexed with guanosine-5'-(beta,gamma-imido) triphosphate and guanosine-5'-(beta,gamma-methylene)triphosphate in the presence and the absence of Mg2+ have been refined to R-factors below 0.2 against data to a nominal resolution of 2.7 A. Asp333 of the synthetase hydrogen bonds to the exocyclic 2-amino and endocyclic N1 groups of the guanine nucleotide base, whereas the hydroxyl of Ser414 and the backbone amide of Lys331 hydrogen bond to the 6-oxo position. The side chains of Lys331 and Pro417 pack against opposite faces of the guanine nucleotide base. The synthetase recognizes neither the N7 position of guanine nucleotides nor the ribose group. Electron density for the guanine-5'-(beta,gamma-imido) triphosphate complex is consistent with a mixture of the triphosphate nucleoside and its hydrolyzed diphosphate nucleoside bound to the active site. The base, ribose, and alpha-phosphate positions overlap, but the beta-phosphates occupy different binding sites. The binding of guanosine-5'-(beta,gamma-methylene)triphosphate to the active site is comparable with that of guanosine-5'-(beta, gamma-imido)triphosphate. No electron density, however, for the corresponding diphosphate nucleoside is observed. In addition, electron density for bound Mg2+ is absent in these nucleotide complexes. The guanine nucleotide complexes of the synthetase are compared with complexes of other GTP-binding proteins and to a preliminary structure of the complex of GDP, IMP, Mg2+, and succinate with the synthetase. The enzyme, under conditions reported here, does not undergo a conformational change in response to the binding of guanine nucleotides, and minimally IMP and/or Mg2+ must be present in order to facilitate the complete recognition of the guanine nucleotide by the synthetase.     

Expression of mRNA encoding IFN alpha in macrophages stimulated with Lactobacillus gasseri

The N7 of guanine is thought to be the primary target for adduct and crosslink formation between cisplatin and DNA. However, reactive sites in DNA other than the N7 of guanine may also participate in the formation of adducts with cisplatin. The possibility that these interactions arise and form DNA polymerase blocking lesions was investigated by primer extension reactions with Taq DNA polymerase. To differentiate between damage produced at relatively weak sites from those formed at the N7 of guanine, a modified DNA template was synthesised with the N7 of guanine replaced with a carbon atom. This was achieved in a PCR designed to incorporate 7-deazaguanine instead of normal guanine. The sequence specificity of cisplatin damage in the modified and unmodified DNA substrates was compared (after linear amplification) by DNA sequencing gel analysis. For concentrations of cisplatin (1 to 5 microM) that induce blocking lesions in normal DNA, no significant damage was observed in the modified DNA. This confirmed that the N7 of guanine is the major site of adduct formation in normal DNA. At higher concentrations of cisplatin (50 microM and 100 microM), lesions were found at AA dinucleotides and other novel sites in the modified DNA. These results indicate that the N7 of guanine is not required in the formation of some cisplatin adducts.     

Long-range and short-range oxidative damage to DNA: photoinduced damage to guanines in ethidium-DNA assemblies

Short-range and long-range photoreactions between ethidium and DNA have been characterized. While no DNA reaction is observed upon excitation into the visible absorption band of ethidium, higher-energy irradiation (313-340 nm) leads both to direct strand cleavage at the 5'-G of 5'-GG-3' doublets and to piperidine-sensitive lesions at guanine. This reactivity is not consistent with oxidation of guanine by either electron transfer or singlet oxygen as shown by comparison with reactions of a rhodium intercalator and methylene blue, respectively. By covalently tethering ethidium to one end of a DNA duplex, we demonstrate the presence of two distinct reactions, one short-range and the other long-range. The short-range reaction involves a covalent modification of guanine by ethidium, based upon HPLC analysis of the nucleoside products and studies with ethidium derivatives. The long-range reaction is entirely consistent with oxidation of guanine by DNA-mediated electron transfer. The yield of this electron-transfer reaction is not attenuated with distance; equal yields of guanine damage are observed at a proximal (17 A Et-GG separation) and distal (44 A Et-GG separation) site. These results are quite similar to those previously observed with a covalently tethered rhodium photooxidant and underscore the unique ability of the DNA base stack to facilitate long-range electron transfer so as to effect oxidative damage from a distance.     

Topoisomerase II-mediated site-directed alkylation of DNA by psorospermin and its use in mapping other topoisomerase II poison binding sites

Psorospermin is a plant natural product that shows significant in vivo activity against P388 mouse leukemia. The molecular basis for this selectivity is unknown, although psorospermin has been demonstrated to intercalate into DNA and alkylate N7 of guanine. Significantly, the alkylation reactivity of psorospermin at specific sites on DNA increased 25-fold in the presence of topoisomerase II. In addition, psorospermin trapped the topoisomerase II-cleaved complex formation at the same site. These results imply that the efficacy of psorospermin is related to its interaction with the topoisomerase II-DNA complex. Because thermal treatment of (N7 guanine)-DNA adducts leads to DNA strand breakage, we were able to determine the site of alkylation of psorospermin within the topoisomerase II gate site and infer that intercalation takes place at the gate site between base pairs at the +1 and +2 positions. These results provide not only additional mechanistic information on the mode of action of the anticancer agent psorospermin but also structural insights into the design of an additional class of topoisomerase II poisons. Because the alkylation site for psorospermin in the presence of topoisomerase II can be assigned unambiguously and the intercalation site inferred, this drug is a useful probe for other topoisomerase poisons where the sites for interaction are less well defined.     

Parallel-stranded duplexes and quartet assemblies formed by oligonucleotides containing isoguanine

Oligonucleotides with isoguanine-cytosine base pairs form duplexes with parallel chain orientation. This strong base pair dictates the strand polarity when additional dA-dT or N7Ad-dT pairs are present. The same is observed for the isocytosine-guanine pair. Quartet structures are built by the self-assembly of oligonucleotides containing short runs of isoguanine or 7-deazaisoguanine. New base pairs between isoguanine or guanine and 5-aza-7-deazaguanine are presented.     

A cyclic N7,C-8 guanine adduct of N-nitrosopyrrolidine (NPYR): formation in nucleic acids and excretion in the urine of NPYR-treated rats

N-Nitrosopyrrolidine (NPYR) is a well-established hepatocarcinogen that is present in the diet and tobacco smoke and may form endogenously in humans. Biomarkers to assess NPYR exposure and metabolic activation in humans are needed. The cyclic N7,C-8 guanine adduct 2-amino-6,7,8,9-tetrahydro-9-hydroxypyrido[2,1-f]purin-4(3H)-one (8), which is formed in tissues of rats treated with NPYR, is one potential candidate for such a biomarker. In this study, we evaluated the formation of this and other NPYR adducts in reactions of alpha-acetoxyNPYR with dGuo, Guo, DNA, and RNA and determined the extent of urinary excretion of adduct 8 in rats treated with NPYR. alpha-AcetoxyNPYR, a stable precursor to the major product of NPYR metabolic activation, was allowed to react with dGuo, Guo, DNA, or RNA at 37 degrees C, pH 7. The most striking observation was that the cyclic N7,C-8 guanine adduct 8 was formed 9 times more extensively in the reaction with Guo than with dGuo. It was also formed 2.5 times more extensively in RNA than in DNA. In rats treated with NPYR, levels of the cyclic N7,C-8 guanine adduct 8 were 2 times as high in RNA than in DNA. Rats treated with [14C]adduct 8 excreted 51% of this adduct unchanged in urine. Rats treated with [3,4-3H]NPYR excreted 0.00004% of the dose as adduct 8. The major differences in product formation in reactions of alpha-acetoxyNPYR with dGuo versus Guo are unusual for alkylating agents; potential mechanisms are discussed. The higher levels of adduct 8 in RNA than in DNA suggest that RNA may be superior as a source of adduct 8 as a biomarker.     

Reaction of aflatoxin B1 exo-8,9-epoxide with DNA: kinetic analysis of covalent binding and DNA-induced hydrolysis

The exo isomer of aflatoxin B1 (AFB1) 8,9-epoxide appears to be the only product of AFB1 involved in reaction with DNA and reacts with the N7 atom of guanine via an SN2 reaction from an intercalated state. Although the epoxide hydrolyzes rapidly in H2O (0.6 s-1 at 25 degrees C), very high yields of DNA adduct result. Experimental binding data were fit to a model in which the epoxide forms a reversible complex with calf thymus DNA (Kd = 0.43 mg ml-1, or 1.4 mM monomer equivalents) and reacts with guanine with a rate of 35 s-1. Stopped-flow kinetic analysis revealed attenuation of fluorescence in the presence of DNA that was dependent on DNA concentration. Kinetic spectral analysis revealed that this process represents conjugation of epoxide with DNA, with an extrapolated rate maximum of 42 s-1 and half-maximal velocity at a DNA concentration of 1.8 mg ml-1 (5.8 mM monomer equivalents). The rate of hydrolysis of the epoxide was accelerated by calf thymus DNA in the range of pH 6-8, with a larger enhancement at the lower pH (increase of 0.23 s-1 at pH 6.2 with 0.17 mg DNA ml-1). The same rate enhancement effect was observed with poly[dA-dT].poly[dA-dT], in which the epoxide can intercalate but not form significant levels of N7 purine adducts, and with single-stranded DNA. The increased rate of hydrolysis by DNA resembles that reported earlier for epoxides of polycyclic hydrocarbons and is postulated to involve a previously suggested localized proton field on the periphery of DNA. The epoxide preferentially intercalates between base pairs, and the proton field is postulated to provide acid catalysis to the conjugation reaction.     

Guanine is the target for direct ionisation damage in DNA, as detected using excision enzymes

Exposure of an aqueous, aerated solution (pH 7) of a double-stranded DNA to 193 nm light, of sufficient energy to ionise DNA, leads to selective, non-random modification at guanine in the form of frank single-strand break (ssb) and base modifications, revealed by treatment with either Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg), Escherichia coli endonuclease III (Nth) or hot piperidine treatment. There is a similar neighbouring base sequence dependence for Fpg- and Nth-sensitive damage as that previously reported for both hot alkali-labile damage and prompt ssb. Low yields of photoproducts, namely pyrimidine dimers, are also revealed using the enzyme T4 endonuclease V (T4 endo V). Although irradiation of DNA with 193 nm light causes photoionisation of all the nucleic acid bases, these results indicate that guanine is the predominant site for localisation of the oxidative damage. These findings are consistent with migration of the radical cation to 'target' damage at guanine sites.     

Polymerase recognition of synthetic oligodeoxyribonucleotides incorporating degenerate pyrimidine and purine bases

A universal base that is capable of substituting for any of the four natural bases in DNA would be of great utility in both mutagenesis and recombinant DNA experiments. This paper describes the properties of oligonucleotides incorporating two degenerate bases, the pyrimidine base 6H,8H-3,4-dihydropyrimido[4,5-c][1,2]oxazin-7-one and the purine base N6-methoxy-2,6-diaminopurine, designated P and K, respectively. An equimolar mixture of the analogues P and K (called M) acts, in primers, as a universal base. The thermal stability of oligonucleotide duplexes were only slightly reduced when natural bases were replaced by P or K. Templates containing the modified bases were copied by Taq polymerase; P behaved as thymine in 60% of copying events and as cytosine in 40%, whereas K behaved as if it were guanine (13%) or adenine (87%). The dUTPase gene of Caenorhabditis elegans, which we have found to contain three nonidentical homologous repeats, was used as a model system to test the use of these bases in primers for DNA synthesis. A pair of oligodeoxyribonucleotides, each 20 residues long and containing an equimolar mixture of P and K at six positions, primed with high specificity both T7 DNA polymerase in sequencing reactions and Taq polymerase in PCRs; no nonspecific amplification was obtained on genomic DNA of C. elegans. Use of P and K can significantly reduce the complexity of degenerate oligonucleotide mixtures, and when used together, P and K can act as a universal base.     

Effect of dietary restriction on benzo[a]pyrene (BaP) metabolic activation and pulmonary BaP-DNA adduct formation in mouse

Hepatic microsomal xenobiotic metabolizing enzyme activities of laboratory animals can be modulated by Dietary restriction (DR). The modulation of xenobiotic metabolizing enzyme activities can affect the metabolic activation of chemical carcinogens. Acute DR (60% of the food consumption of ad libitum (AL)-fed mice for 7 weeks) reduced the body weights of the male B6C3F1 mice, and increased mouse pulmonary cytochrome P4501A1-dependent BaP metabolizing enzyme activity. The effects of DR on the formation of the specific BaP-DNA adduct, 10-(N2-deoxyguanosinyl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-BaP (BaP-N2-dG) in mouse lung can be detected by using 32P-postlabeling technique. In both AL- and DR-mice total BaP-DNA adduct formation in lung reached a peak at 48 hours after treatment with [3H]BaP and the in vivo formation of BaP-N2-dG was greater in DR mouse lung than in that of AL-animals by 22%. DR increased in vitro BaP-N2-dG formation by 39% when calf-thymus DNA was incubated with BaP using liver microsomes obtained from DR- or AL-mice as the enzyme source. The formation of the specific BaP-N2-dG adducts, measured by 32P-postlabeling, was only 20% of the total [3H]BaP-DNA adducts as determined by liquid scintillation counting. The increase of BaP-DNA adduct formation in mouse lung was correlated to the enhancement of the mouse pulmonary BaP metabolizing enzyme activity. Our results indicated that the effect of DR on the metabolic activation of BaP in mouse lung was dependent upon the mouse lung cytochrome P4501A1-dependent BaP metabolizing enzymes activities which was significantly increased by DR.     

DNA repair in congenic mice: possible influence of a chromosome 4 genetic region on the rate of benzo[a]pyrene-induced DNA adduct removal

An attempt was made to assign mouse lifespan-associated interstrain differences in DNA repair to a specific chromosomal region using a set of congenic mice. The sensitive 32P-postlabeling assay was employed to measure the removal of benzo[a]pyrene-induced DNA adducts in liver DNA of three different chromosome 4 congenic mouse strains: B6.C-H-15c, B6.C-H-16c, and B6.C-H-26c and the two parental strains, C57B1/6 and BALB/c. The removal of the one main adduct detected, trans-(7R)-N2-[10-(7 beta,8 alpha,9 alpha-trihydroxy)-7,8,9,10- tetrahydrobenzo(a)-pyrene]-yl-deoxyguanosine (BPDE-N2-dG), in liver DNA of C57Bl/6 and BALB/c mice between one and three days after treatment, was approximately 86% and 57%, respectively. The percentage removal of BPDE-N2-dG in two of the three congenic mouse strains, B6.C-H-16c and B6.C-H-26c, resembled that found in BALB/c, whereas the third strain, B6.C-H-15c, removed about the same amount as C57B1/6, i.e., approximately 88% of BPDE-N2-dG between one and three days after treatment. The usefulness of congenic mouse strains for identifying genes putatively involved in aging and/or disease susceptibility is discussed.     

DNA interactions of a novel platinum drug, cis-[PtCl(NH3)2(N7-acyclovir)]+

We synthesized a novel platinum drug, cis-[PtCl(NH3)2(N7-ACV)]+, in which ACV is the antiviral drug acyclovir [a deoxyriboguanosine analogue, 9-(2-hydroxyethoxymethyl)guanine]. This new compound exhibits antiviral efficacy in vitro and exhibits an antitumor activity profile different from that of cisplatin [Metal-Based Drugs 2:249-256 (1995)]. To contribute to understanding the mechanisms underlying biological activity of this new compound, we studied modifications of natural and synthetic DNAs in cell-free media by cis-[PtCl(NH3)2(N7-ACV)]+ by various biochemical and biophysical methods. The results indicated that the major DNA adduct of cis-[PtCl(NH3)2(N7-ACV)]+ was a stable monofunctional adduct at guanine residues. In contrast to DNA adducts of other monodentate and clinically ineffective platinum(II) compounds, the adducts of cis-[PtCl(NH3)2(N7-ACV)]+ terminated in vitro DNA and RNA synthesis. In addition, although DNA adducts of cis-[PtCl(NH3)2(N7-ACV)]+ and cisplatin were different, some properties of DNA modified by either compound were qualitatively similar. Such similarities were not noticed if DNA modifications by other ineffective monofunctional platinum(II) complexes were investigated. Thus, the DNA binding mode of monofunctional cis-[PtCl(NH3)2(N7-ACV)]+ was different from that of other monofunctional but ineffective platinum(II) complexes. It has been suggested that the unique capability of cis-[PtCl(NH3)2(N7-ACV)]+ to modify DNA may be relevant to a distinct antitumor efficiency of this novel drug in comparison with cisplatin. It also has been suggested that at least some aspects of DNA interactions of cis-[PtCl(NH3)2(ACV)]+ revealed in the current study could be exploited in the search for and development of new antiviral platinum complexes containing, as a part of the coordination sphere, antiviral nucleosides.     

Detection of the sites of alkylation in DNA and polynucleotides by laser Raman spectroscopy

A laser Raman study of the alkylation of calf thymus DNA, poly(dG)-poly(dC) and poly(dA)-(dT) has been made using two water soluble alkylating agents: an antitumor drug, the difunctional methyl nitrogen mustard (HN2), which froms interstrand cross-links, and the dimethyl nitrogen half mustard (HN1). When an excess of the alkylating agent was used, the observed Raman frequencies due to the guanine ring modes in DNA and poly(dG)-poly(dC) changed virtually quantitatively to those of 7-methylguanosine (7-Me-Guo) showing that essentially all of the guanine bases were alkylated in the N-7 position. Furthermore, this alkylated DNA formed a stable double helical complex at neutral pH in which the alkylated guanine residues are in the keto form. No changes in the Raman bands of any of the other bases were observed in alkylated DNA. The DNA double helix, completely alkylated in at the N-7 position of guanine, melts about 35 degrees C below that of the native DNA. Upon melting, the alkylated guanine changes from the keto to the zwitterionic form.     

Mechanisms of DNA damage by chromium(V) carcinogens

Reactions of bis(2-ethyl-2-hydroxy-butanato)oxochromate(V) with pUC19 DNA, single-stranded calf thymus DNA (ss-ctDNA), a synthetic oligonucleotide, 5'-GATCTATGGACTTACTTCAAGGCCGGGTAATGCTA-3' (35mer), deoxyguanosine and guanine were carried out in Bis-Tris buffer at pH 7.0. The plasmid DNA was only nicked, whereas the single-stranded DNA suffered extensive damage due to oxidation of the ribose moiety. The primary oxidation product was characterized as 5-methylene-2-furanone. Although all four bases (A, C, G and T) were released during the oxidation process, the concentration of guanine exceeds the other three. Orthophosphate and 3'-phosphates were also detected in this reaction. Likewise, the synthetic oliogomer exhibits cleavage at all bases with a higher frequecncy at G sites. This increased cleavage at G sites was more apparent after treating the primary oxidation products with piperidine, which may indicate base oxidation as well. DNA oxidation is shown to proceed through a Cr(V)-DNA intermediate in which chromium(V) is coordinated through the phosphodiester moiety. Two alternative mechanisms for DNA oxidation by oxochromate(V) are proposed to account for formation of 5-methylene-2-furanone, based on hydrogen abstraction or hydride transfer from the C1' site of the ribose followed by hydration and two successive beta-eliminations. It appears that phosphate coordination is a prerequisite for DNA oxidation, since no reactions between chromium(V) and deoxyguanosine or guanine were observed. Two other additional pathways, hydrogen abstraction from C4' and guanine base oxidation, are also discussed.     

Refined solution structure of 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 opposite CpA in the complementary strand of an oligodeoxynucleotide duplex as determined by 1H NMR

The solution structure of d(CCATCAFBGATCC).d(GGATCAGATGG), containing the 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 adduct, was refined using molecular dynamics restrained by NOE data obtained from 1H NMR. The modified guanosine was positioned opposite cytosine, while the aflatoxin moiety was positioned opposite adenosine in the complementary strand. Sequential 1H NOEs were interrupted between C5 and AFBG6, but intrastrand NOEs were traced through the aflatoxin moiety, via H6a of aflatoxin and H8 of the modified guanine. Opposite the lesion, the NOE between A16 H1' and G17 H8 was weak. A total of 43 NOEs were observed between DNA protons and aflatoxin protons. Molecular dynamics calculations restrained with 259 experimental and empirical distances, and using sp2 hybridization at AFBG6 N7, refined structures with pairwise rms differences < 0.85 A, excluding terminal base pairs. Relaxation matrix calculations yielded a sixth root rms difference between refined structures and NOE intensity data of 7.3 x 10(-2). The aflatoxin moiety intercalated on the 5'-face of the modified guanine. The extra adenine A16 was inserted between base pair AFBG6.C15 and the aflatoxin moiety. A 36 degree bending between the plane of base pair AFBG6.C15 and the plane of the aflatoxin moiety was predicted. The aflatoxin moiety stacked below the top domain of the oligodeoxynucleotide, which consisted of base pairs C1.G21, C2.G20, A3.T19, T4.A18, and C5.G17. The bottom domain consisted of base pairs AFBG6.C15, A7.T14, T8.A13, C9.G12, and C10.G11. The average winding angle between base pair C5.G17, the intercalated aflatoxin moiety, A16, and base pair AFBG6.C15 was reduced to 10 degrees. The preponderance of base pair substitutions in the aflatoxin B1 mutational spectrum, particularly G-->T transversions, suggests that the stability of this modified oligodeoxynucleotide, which models a templated +1 addition mutation, does not reliably predict the frequency of frame shifts.     

Dissection of the sequence specificity of the Holliday junction endonuclease CCE1

CCE1 is a Holliday (four-way DNA) junction-specific endonuclease which resolves mitochondrial DNA recombination intermediates in Saccharomycescerevisiae. The junction-resolving enzymes are a diverse class, widely distributed in nature from viruses to higher eukaryotes. In common with most other junction-resolving enzymes, the cleavage activity of CCE1 is nucleotide sequence-dependent. We have undertaken a systematic study of the sequence specificity of CCE1, using a single-turnover kinetic assay and a panel of synthetic four-way DNA junction substrates. A tetranucleotide consensus cleavage sequence 5'-ACT downward arrowA has been identified, with specificity residing mainly at the central CT dinucleotide. Equilibrium constants for CCE1 binding to four-way junctions are unaffected by sequence variations, suggesting that substrate discrimination occurs predominantly in the transition state complex. CCE1 cuts most efficiently at the junction center, but can also cleave the DNA backbone at positions one nucleotide 3' or 5' of the point of strand exchange, suggesting a significant degree of conformational flexibility in the CCE1:junction complex. Introduction of base analogues at single sites in four-way junctions has allowed investigation of the sequence specificity of CCE1 in finer detail. In particular, the N7 moiety of the guanine base-pairing with the cytosine of the consensus sequence appears to be crucial for catalysis. The functional significance of sequence specificity in junction-resolving enzymes is discussed.     

Radiation chemistry of d(ApCpGpT)

The radiation chemistry of the DNA tetranucleoside triphosphate d(ApCpGpT) was investigated. Irradiations were carried out on aqueous solutions saturated with oxygen (with and without added Cu++), nitrogen or nitrous oxide. When oxygen was present, principal products were formed by hydroxylation at the 8-position of guanine and by degradation of thymine leaving a formamido remnant. Products were also formed containing both of the aforementioned lesions at adjacent deoxyguanosine and pyrimidine nucleosides. Other products resulted from rearrangement of the thymine ring generating two diastereoisomers of the 5-methyl-5-hydroxyhydantoin modification of d(ApCpGpT). Rearrangement of the cytosine ring occurred generating imidazolidine products and a hydantoin product. The product profiles are similar when either an N2O or N2 gaseous environment is maintained. However, in the latter case the dihydrothymine modifications of d(ApCpGpT) are markedly enhanced. Other products include an 8,5' cyclized product formed from the 2'-deoxyadenosine nucleoside and the 8-hydroxyguanine modification. 6-Hydroxy-5,6-dihydrothymine and 5,6-dihydroxy-5,6-dihydrothymine modifications of the thymidine nucleoside were also observed. A strand break product formed in oxygenated solution is also produced in nitrous oxide saturated solutions. Scission of the deoxyadenosine terminus was also observed. The effect of several of these lesions on d(ApCpGpT) as substrate for nuclease P1, bovine spleen phosphodiesterase and snake venom phosphodiesterase was studied.     

Structures of free and complexed forms of Escherichia coli xanthine-guanine phosphoribosyltransferase

Structures of free, substrate-bound and product-bound forms of Escherichia coli xanthine-guanine phosphoribosyltransferase (XGPRT) have been determined by X-ray crystallography. These are compared with the previously determined structure of magnesium and sulphate-bound XPRT. The structure of free XGPRT at 2.25 A resolution confirms the flexibility of residues in and around a mobile loop identified in other PRTases and shows that the cis-peptide conformation of Arg37 at the active site is maintained in the absence of bound ligands. The structures of XGPRT complexed with the purine base substrates guanine or xanthine in combination with cPRib-PP, an analog of the second substrate PRib-PP, have been solved to 2.0 A resolution. In these two structures the disordered phosphate-binding loop of uncomplexed XGPRT becomes ordered through interactions with the 5'-phosphate group of cPRib-PP. The cyclopentane ring of cPRib-PP has the C3 exo pucker conformation, stabilised by the cPRib-PP-bound Mg2+. The purine base specificity of XGPRT appears to be due to water-mediated interactions between the 2-exocyclic groups of guanine or xanthine and side-chains of Glu136 and Asp140, as well as the main-chain oxygen atom of Ile135. Asp92, together with Lys115, could help stabilise the N7-protonated tautomer of the incoming base and could act as a general base to remove the proton from N7 when the nucleotide product is formed. The 2.6 A resolution structure of XGPRT complexed with product GMP is similar to the substrate-bound complexes. However, the ribose ring of GMP is rotated by approximately 24 degrees compared with the equivalent ring in cPRib-PP. This rotation results in the loss of all interactions between the ribosyl group and the enzyme in the product complex.