Sequence selectivity of azinomycin B in DNA alkylation and cross-linking: a QM/MM study

Azinomycin B—a well-known antitumor drug—forms cross-links with DNA through alkylation of purine bases and blocks tumor cell growth. This reaction has been modeled using the ONIOM (B3LYP/6-31?+?g(d):UFF) method to understand the mechanism and sequence selectivity. ONIOM results have been checked for reliability by comparing them with full quantum mechanics calculations for selected paths. Calculations reveal that, among the purine bases, guanine is more reactive and is alkylated by aziridine ring through the C10 position, followed by alkylation of the epoxide ring through the C21 position of Azinomycin B. While the mono alkylation is controlled kinetically, bis-alkylation is controlled thermodynamically. Solvent effects were included using polarized-continuum-model calculations and no significant change from gas phase results was observed.

Characterization of AziR, a resistance protein of the DNA cross-linking agent azinomycin B

A protein identified from the Streptomyces sahachiroi genome exhibits a protective effect against the DNA alkylator azinomycin B when heterologously expressed in S. lividans and E. coli. The protein, dubbed AziR for azinomycin resistance, is homologous to aminoglycoside phosphotransferases but behaves as an azinomycin binding protein and fails to chemically modify azinomycin. While AziR confers resistance to azinomycin B, it is inactive against aminoglycoside antibiotics and other DNA alkylators. A nucleic acid staining assay indicates that the protein enhances cell survival, and also prevents DNA damage effects normally observed following azinomycin treatment. Knowledge of an azinomycin resistance mechanism aids in setting the stage for future engineered biosynthesis of functionally useful azinomycin analogues.     

Orientation of the Lac repressor DNA binding domain in complex with the left lac operator half site characterized by affinity cleaving.

Lac repressor (LacR) is a helix-turn-helix motif sequence-specific DNA binding protein. Based on proton NMR spectroscopic investigations, Kaptein and co-workers have proposed that the helix-turn-helix motif of LacR binds to DNA in an orientation opposite to that of the helix-turn-helix motifs of lambda repressor, lambda cro, 434 repressor, 434 cro, and CAP [Boelens, R., Scheek, R., van Boom, J. and Kaptein, R., J. Mol. Biol. 193, 1987, 213-216]. In the present work, we have determined the orientation of the helix-turn-helix motif of LacR in the LacR-DNA complex by the affinity cleaving method. The DNA cleaving moiety EDTA.Fe was attached to the N-terminus of a 56-residue synthetic protein corresponding to the DNA binding domain of LacR. We have formed the complex between the modified protein and the left DNA half site for LacR. The locations of the resulting DNA cleavage positions relative to the left DNA half site provide strong support for the proposal of Kaptein and co-workers.     

Fluorescence study of DNA alkylation by epoxides

A simple fluorescence assay was devised to measure alkylation of guanine. The assay was tested with simple epoxides: propylene oxide, glycidol, epichlorohydrin, trichloropropylene oxide and styrene oxide, which are known to vary considerably in their mutagenic potency. The order of reactivity parallelled the mutagenic potency, trichloropropylene oxide being the most reactive alkylating agent. Each epoxide alkylated deoxyguanosine faster than single-stranded DNA, at equal concentrations of guanine. Single-stranded DNA was alkylated substantially faster than was double-stranded DNA. The reaction products with each substrate were analysed by thin-layer chromatography and exhibited similar Rf-values. It was concluded that polymers, particularly double-stranded DNA, reacted slower than deoxyguanosine due to the properties of polymers in solution rather than the unavailability of reactive sites for alkylation.     

Mechanism of kappa B DNA binding by Rel/NF-kappa B dimers

The DNA binding of three different NF-kappaB dimers, the p50 and p65 homodimers and the p50/p65 heterodimer, has been examined using a combination of gel mobility shift and fluorescence anisotropy assays. The NF-kappaB p50/p65 heterodimer is shown here to bind the kappaB DNA target site of the immunoglobulin kappa enhancer (Ig-kappaB) with an affinity of approximately 10 nm. The p50 and p65 homodimers bind to the same site with roughly 5- and 15-fold lower affinity, respectively. The nature of the binding isotherms indicates a cooperative mode of binding for all three dimers to the DNA targets. We have further characterized the role of pH, salt, and temperature on the formation of the p50/p65 heterodimer-Ig-kappaB complex. The heterodimer binds to the Ig-kappaB DNA target in a pH-dependent manner, with the highest affinity between pH 7.0 and 7.5. A strong salt-dependent interaction between Ig-kappaB and the p50/p65 heterodimer is observed, with optimum binding occurring at monovalent salt concentrations below 75 mm, with binding becoming virtually nonspecific at a salt concentration of 200 mm. Binding of the heterodimer to DNA was unchanged across a temperature range between 4 degrees C and 42 degrees C. The sensitivity to ionic environment and insensitivity to temperature indicate that NF-kappaB p50/p65 heterodimers form complexes with specific DNA in an entropically driven manner.     

In vitro DNA and nuclear proteins alkylation by 1,2-dimethylhydrazine

The methylating carcinogen 1,2-dimethylhydrazine (DMH) CAS 540.73.8 is highly organ-specific and, under certain experimental conditions, produces a high incidence of adenocarcinoma in the colon of rodents. We have tried to assess the possibility that part of the organ-specifity in the carcinogenic effect of DMH could be attributed to its metabolism by specific microsomal enzymes. In particular, we compared the in vitro effects of DMH in the presence of either colon or liver microsomes from animals that had been treated with microsomal enzyme inducers. V79 Chinese hamster cells were used as the target to evaluate the damage to the genetic material, as judged by (1) formation of adducts of DNA bases and (2) amino acid modifications in nuclear proteins using [Me-14C]DMH and appropriate analytical detection systems. Our results tend to support the above postulated hypothesis.     

Effects of alkylation by dimethyl sulfate, nitrogen mustard, and mitomycin C on DNA structure as studied by the ethidium binding assay.

The extent of alkylation of DNA by dimethyl sulfate, nitrogen mustard, and the antibiotic mitomycin C is related to the resulting decrease in the fluorescence of intercalated ethidium. The fluorescence losses due to the first two types of reagents show a marked pH dependence, with greater losses of fluorescence being observed at alkaline pH values. At pH 11.6 the fluorescence shows a slow recovery, so that with low levels of methylation (4% deoxyguanosine residues modified) one observes complete return of fluorescence. We postulate that these phenomena are due to conversion of 7-methyldeoxyguanosine to the zwitterionic form, and partial denaturation of the DNA duplex with loss of ethidium binding sites. Hydroxide-ion-catalyzed imidazole ring opening, and the removal of the positive charge permits reannealing with concomitant return of the ethidium intercalation sites. This conclusion is substantiated by enzymatic hydrolysis of 14C-labelled methylated DNA and identifiions of the ethidium assay. The distinctly different behavior of mitomycin C confirms previous conclusions that its alkylation, preferentially on guanine, does not take part at the N-7 position.     

Mechanism of monofunctional and bifunctional alkylation of DNA by mitomycin C

The relative amounts of monofunctional and bifunctional alkylation products of DNA with mitomycin C (MC) depend on whether one or both masked alkylating functions of MC are activated reductively; adduct 8 is the result of one function and adducts 7 and 9, formed as a pair, are the result of both functions being activated [Tomasz, M., Lipman, R., Chowdary, C., Pawlak, J., Verdine, G. L., & Nakanishi, K. (1987) Science (Washington, D.C.) 235, 1204-1208]. To determine the mechanism governing this differential reactivity of MC with DNA, MC-Micrococcus luteus DNA complexes formed under varying conditions in vitro were digested to nucleosides and adducts. Adduct distribution, analyzed by high-performance liquid chromatography, served as the measure of monofunctional and bifunctional activation. H2/PtO2 and xanthine oxidase/reduced nicotinamide adenine dinucleotide (NADH) activated MC mostly monofunctionally, and Na2S2O4 activated the drug bifunctionally under comparable conditions. Excess MC selectively suppressed, but excess PtO2 selectively promoted, bifunctional activation by H2/PtO2; excess xanthine oxidase and/or NADH also had promoting effects. O2 tested in the Na2S2O4 system was inhibitory. 10-Decarbamoyl-MC acted strictly monofunctionally under all conditions. Monoadducts bound to DNA were converted to bis adducts upon rereduction. A mechanism with the following features was derived: (i) Activation of MC at C-1 and C-10 is sequential (C-1 first). (ii) A one-time reduction is sufficient for both. (iii) Activation of the second function may be selectively inhibited by kinetic factors or O2. (iv) 7 and 9 are coproducts of bifunctional activation; their ratio depends on the DNA base sequence. (v) Activation of the second function involves an iminium intermediate. Direct applications to the action of MC in vivo are discussed.     

Site-specific and photo-induced alkylation of DNA by a dimethylanthraquinone-oligodeoxynucleotide conjugate.

A dialkyl-substituted anthraquinone derivative was synthesized and ligated to a sequence-directing oligodeoxynucleotide to examine its efficiency and specificity for cross-linking to complementary sequences of DNA. The anthraquinone appendage stabilized spontaneous hybridization of the target and probe sequences through non-covalent interactions, as indicated by thermal denaturation studies. Covalent modification of the target was induced by exposure to near UV light (lambda > 335 nm) to generate cross-linked duplexes in yields as great as 45%. Reaction was dependent on the first unpaired nucleotide extended beyond the duplex formed by association of the target and probe. A specificity of C > T > A = G was determined for modification at this position. The overall site and nucleotide selectivity seems to originate from the chemical requirements of cross-linking and does not likely reflect the dominant solution structure of the complex prior to irradiation.     

DNA alkylation by leinamycin can be triggered by cyanide and phosphines.

Previous work has shown that alkylation of DNA by the antitumor agent leinamycin (1) is potentiated by reaction of the antibiotic with thiols. Here, it is shown that other soft nucleophiles such as cyanide and phosphines can also trigger DNA alkylation by leinamycin. Overall, the results suggest that reactions of cyanide and phosphines with leinamycin produce the oxathiolanone intermediate (2), which is known to undergo rearrangement to the DNA-alkylating episulfonium ion 4.     

Molecular basis for sequence-specific DNA alkylation by CC-1065

CC-1065 is a potent antitumor antibiotic that binds covalently to N3 of adenine in the minor groove of DNA. The CC-1065 molecule is made up of three repeating pyrroloindole subunits, one of which (the left-hand one or A subunit) contains a reactive cyclopropyl function. The drug reacts with adenines in DNA in a highly sequence-specific manner, overlapping four base pairs to the 5'-side of the covalently modified base. Concomitant with CC-1065 covalent binding to DNA is an asymmetric effect on local DNA structure which extends more than one helix turn to the 5'-side of the covalent binding site. The DNA alkylation, sequence specificity, and biological potency of CC-1065 and a select group of trimeric synthetic analogues were evaluated. The results suggest that (a) noncovalent interactions between this series of compounds and DNA do not lead to the formation of complexes stable enough to be detected by footprinting methods, (b) sequence specificity and alkylation intensity can be modulated by the substituents on the nonreactive middle and right-hand segments, and (c) biological potency correlates well with ability to alkylate DNA. In addition, the extent and the sequence specificity of covalent adduct formation between linear DNA fragments and three analogues comprised of the CC-1065 alkylating subunit linked to zero (analogue A), one (analogue AB), or two (analogue ABC) nonreactive indole subunits were compared.(ABSTRACT TRUNCATED AT 250 WORDS)     

CAP binding to B and Z forms of DNA.

We have examined the interaction between the cyclic AMP receptor protein (CAP) and a small DNA fragment containing its specific recognition sequence by circular dichroism spectroscopy. The binding of CAP to this fragment induces a B to "C-like" change in the CD spectrum, which is different from that observed for non-specific binding. A one-to-one (CAP dimer to DNA) binding stoichiometry was deduced from spectroscopic titration data, as was a non-specific binding site size of 17 bp/dimer. In addition, we have compared the non-specific binding affinity of CAP for the B and Z forms of synthetic DNA copolymers. A slight preference for the B form was found. These results do not support the recent specific suggestion that CAP binds to a left-handed form of DNA (1), but indicate more generally that an optically detectable conformational change takes place in DNA on binding CAP.     

DNA sequence has an effect on the extent and kinds of alkylation of DNA by a potent carcinogen

A system has been developed to study the effects of base sequence (neighboring bases) upon the alkylation of guanine (G) and adenine (A) bases in DNA. The study was performed on the synthetic polydeoxyribonucleotides, poly(dG).poly(dC), poly(dG-dC).poly(dG-dC), poly(dA).poly(dT), poly(dA-dT).poly(dA-dT), poly(dA-dC).poly(dG-dT), poly(dA-dG).poly(dC-dT), as well as calf thymus DNA. Each polynucleotide was treated with N-[3H]methyl-N-nitrosourea (MNU), depurinated, and the freed alkylpurines separated by HPLC and quantitated by liquid scintillation counting. The amounts of 3-methylguanine (3-MG), 7-MG, and O6-MG relative to guanine, and 3-methyladenine (3-MA) and 1-MA plus 7-MA relative to adenine, and also the O6-MG/7-MG ratios were highly reproducible for a given polynucleotide. Significant differences were found in the amounts of each of the methylpurines formed when compared among the six synthetic polynucleotides and DNA. This evidence is interpreted as an effect upon alkylation which is ultimately dependent upon the base sequence. These findings may have significance in defining the specificity of chemical carcinogens in terms of the susceptability to modification of nucleotide sequences such as those found in certain oncogenes.     

Sequence-specific DNA binding by the MspI DNA methyltransferase.

The MspI methyltransferase (M.MspI) recognizes the sequence CCGG and catalyzes the formation of 5-methylcytosine at the fist C-residue. We have investigated the sequence-specific DNA-binding properties of M.MspI under equilibrium conditions, using gel-mobility shift assays and DNasel footprinting. M.MspI binds to DNA in a sequence-specific manner either alone or in the presence of the normal methyl donor S-adenosyl-L-methionine as well as the analogues, sinefungin and S-adenosyl-L-homocysteine. In the presence of S-adenosyl-L-homocysteine, M.MspI shows the highest binding affinity to DNA containing a hemimethylated recognition sequence (Kd = 3.6 x 10(-7) M), but binds less well to unmethylated DNA (Kd = 8.3 x 10(-7) M). Surprisingly it shows specific, although poor, binding to fully methylated DNA (Kd = 4.2 x 10(-6) M). M.MspI binds approximately 5-fold more tightly to DNA containing its recognition sequence, CCGG, than to nonspecific sequences in the absence of cofactors. In the presence of S-adenosyl-L-methionine, S-adenosyl-L-homocysteine or sinefungin the discrimination between specific and non-specific sequences increases up to 100-fold. DNasel footprinting studies indicate that 16 base pairs of DNA are covered by M.MspI, with the recognition sequence CCGG located asymmetrically within the footprint.     

DNA sequence dependence of guanine-O6 alkylation by the N-nitroso carcinogens N-methyl- and N-ethyl-N-nitrosourea

After intracellular in vitro exposure to the mutagenic and carcinogenic N-nitroso compounds N-methyl-N-nitrosourea (MeNU) or N-ethyl-N-nitrosourea (EtNU), respectively, the average relative amounts of the premutational lesion O⁶-alkylguanine represent about 6% and 8% of all alkylation products formed in genomic DNA. At the level of individual DNA molecules gunine-O⁶ alkylation does nor occur at random; rather, the probability of a substitution reaction at the nucleophilic O⁶ atom is influenced by nucleotide sequence, DNA conformation, and chromatin structure. In the present study, 5 different double-stranded polydeoxynucleotides and 15 double-stranded oligodeoxynucleotides (24-mers) were reacted with MeNU or EtNU in vitro under standardized conditions. Using a competitive radioimmunoassay in conjunction with an anti-(O⁶-2′-deoxyguanosine) monoclonal antibody, the frequency of guanine-O⁶ alkylation was found to be strongly dependent on the nature of the nucleotides flanking guanine on the 5 and 3′ sides. Thus, a 5′ neighboring guanine, followed by 5 adenine and 5′ cytosine, provided an up to 10-fold more ‘permissive’ condition for O⁶-alkylation of the central guanine than a 5′ thymine (with a 5-methylcytocine in the 5′ position being only slightly less inhibitory). Thymine and cytosine were more ‘permissive’ when placed 3′ in comparison with their affects in the 5′ flanking position.