Recognition and repair of 2-aminofluorene- and 2-(acetylamino)fluorene-DNA adducts by UVRABC nuclease

Recognition of damage induced by N-hydroxy-2-aminofluorene (N-OH-AF) and N-acetoxy-2-(acetylamino)fluorene (NAAAF) in both phi X174 RFI supercoiled DNA and a linear DNA fragment by purified UVRA, UVRB, and UVRC proteins was investigated. We have previously demonstrated that N-OH-AF and NAAAF treatments produce N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) and N-(deoxyguanosin-8-yl)-2-(acetylamino)fluorene (dG-C8-AAF), respectively, in DNA. Using a piperidine cleavage method and DNA sequence analysis, we have found that all guanine residues can be modified by N-OH-AF and NAAAF. These two kinds of adducts have different impacts on the DNA helix structure; while dG-C8-AF maintains the anti configuration, dG-C8-AAF is in the syn form. phi X174 RF DNA-Escherichia coli transfection results indicate that while the uvrA, uvrB, and uvrC gene products are needed to repair dG-C8-AAF, the uvrC, but not the uvrA or uvrB gene products, is needed for repair of dG-C8-AF. However, we have found that in vitro the UVRA, UVRB, and UVRC proteins must work in concert to nick both dG-C8-AF and dG-C8-AAF. In general, the reactions of UVRABC nuclease toward dG-C8-AF are similar to those toward dG-C8-AAF; it incises seven to eight nucleotides from the 5' side and three to four nucleotides from the 3' side of the DNA adduct. Evidence is presented to suggest that hydrolysis on the 3' and 5' sides of the damaged base by UVRABC nuclease is not simultaneous and that at least occasionally hydrolysis occurs only on the 3' side or on the 5' side of the damage site. The possible mechanisms of UVRABC nuclease incision for AF-DNA are discussed.     

Construction and characterization of a site-directed CC-1065-N3-adenine adduct within a 117 base pair DNA restriction fragment

The design, construction, and characterization of a site-directed CC-1065-N3-adenine adduct in a 117 base pair segment of M13mpI DNA are described. CC-1065 is an extremely potent antitumor antibiotic produced by Streptomyces zelensis. Previous studies have demonstrated that the cyclopropyl ring of CC-1065 reacts quite specifically with N3 of adenine in double-stranded DNA to form a CC-1065-DNA adduct. Following alkylation, the drug molecule lies snugly within the minor groove of DNA, overlapping with five base pairs for which a marked sequence preference exists [Hurley, L. H., Reynolds, V. R., Swenson, D. H., Petzold, G. L., & Scahill, T. A. (1984) Science (Washington, D.C.) 226, 843-844]. On the basis of the unique characteristics of the reaction of CC-1065 with DNA and the structure of the resulting DNA adduct, we have designed a general strategy to construct a site-directed CC-1065-DNA adduct in a restriction fragment. The presence of unique AluI and HaeIII restriction enzymes sites on each side of a high-affinity CC-1065 binding sequence (5'-GATTA) permitted the preparation of a partial duplex DNA molecule containing the CC-1065 binding sequence in the duplex DNA region. Since CC-1065 only binds to duplex DNA, potential CC-1065 binding sequences in the long single-stranded regions were protected from drug binding during the construction process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differences and similarities in the repair of two benzo[a]pyrene diol epoxide isomers induced DNA adducts by uvrA, uvrB, and uvrC gene products.

We have determined the role of the uvrA, uvrB, and uvrC genes in Escherichia coli cells in repairing DNA damage induced by three benzo[a]pyrene diol epoxide isomers. Using the phi X174 RF DNA-E. coli transfection system, we have found that BPDE-I or BPDE-II modified phi X174 RF DNA has much lower transfectivity in uvrA, uvrB, and uvrC mutant cells compared to wild type cells. In contrast, BPDE-III modification of phi X174 RF DNA causes much less difference in transfectivity between wild type and uvr- mutant cells. Moreover, BPDE-I and -II-DNA adducts are much more genotoxic than are BPDE-III-DNA adducts. Using purified UVRA, UVRB, and UVRC proteins, we have found that these three gene products, working together, incise both BPDE-I- and BPDE-III-DNA adducts quantitatively and, more importantly, at the same rate. In general, UVRABC nuclease incises on both the 5' (six to seven nucleotides) and 3' (four nucleotides) sides of BPDE-DNA adducts with similar efficiency with few exceptions. Quantitation of the UVRABC incision bands indicates that both of these BPDE isomers have different sequence selectivities in DNA binding. These results suggest that although UVR proteins can efficiently repair both BPDE-I- and BPDE-III-DNA adducts, in vivo the uvr system is the major excision mechanism for repairing BPDE-I-DNA adducts but may play a lesser role in repairing BPDE-III-DNA adducts. It is possible the low lethality of BPDE-III-DNA adducts is due to less complete blockage of DNA replication.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequence-dependent interactions of two forms of UvrC with DNA helix-stabilizing CC-1065-N3-adenine adducts

The uvrA, uvrB, and uvrC genes of Escherichia coli control the initial steps of nucleotide excision repair. The uvrC gene product is involved in at least one of the dual incisions produced by the UvrABC complex. Using single-stranded (ss) DNA affinity chromatography, we have separated two forms of UvrC from both wild-type E. coli cells and overproducing cells. UvrCI elutes at 0.4 M KCl, and UvrCII elutes at 0.6 M KCl. In general, both forms, in the presence of UvrA and UvrB, actively incise UV-irradiated and CC-1065-modified DNA in the same fashion; i.e., they incise six to eight nucleotides 5' to and three to five nucleotides 3' to a photoproduct or a CC-1065-N3-adenine adduct. They produce different incisions, however, at a CC-1065-N3-adenine adduct in the sequence 5'-GATTACG- present in the MspI-BstNI 117 bp fragment of M13mp1. UvrABCI incises at both the 5' and 3' sides of the adduct (UvrABCI cut), while UvrABCII incises only at the 5' side (UvrABCII cut). Mixing UvrCI and UvrCII results in both UvrABCI and UvrABCII cuts, and the levels of these two types of cutting are proportional to the amount of UvrCI and UvrCII. DNase I footprints of the MspI-BstNI 117 bp DNA fragment containing a site-directed CC-1065-adenine adduct at the 5'-GATTACG- site show that UvrCII, but not UvrCI, binds to the adduct site. Furthermore, the pattern of DNase I footprints induced by UvrCII binding differs from the pattern of the footprints induced by UvrA, UvrAB, and UvrABCI binding. Interestingly, while the presence of unirradiated DNA enhances the efficiency of UvrABCII in incising UV-irradiated DNA, it does not enhance UvrABCII incision of the CC-1065-N3-adenine adduct formed at 5'-GATTACG-. These results show that two different forms of UvrC differ in DNA binding properties as well as incision modes at some kinds of DNA damage.     

Reaction of the antitumor antibiotic CC-1065 with DNA. Location of the site of thermally induced strand breakage and analysis of DNA sequence specificity

CC-1065 is a unique antitumor antibiotic produced by Streptomyces zelensis. The potent cytotoxic effects of this drug are thought to be due to its ability to form a covalent adduct with DNA through N3 of adenine. Thermal treatment of CC-1065-DNA adducts leads to DNA strand breakage. We have shown that the CC-1065 structural modification of DNA that leads to DNA strand breakage is related to the primary alkylation site on DNA. The thermally induced DNA strand breakage occurs between the deoxyribose at the adenine covalent binding site and the phosphate on the 3' side. No residual modification of DNA is detected on the opposite strand around the CC-1065 lesion. Using the early promoter element of SV40 DNA as a target, we have examined the DNA sequence specificity of CC-1065. A consensus sequence analysis of CC-1065 binding sites on DNA reveals two distinct classes of sequences for which CC-1065 is highly specific, i.e., 5'PuNTTA and 5'AAAAA. The orientation of the DNA sequence specificity relative to the covalent binding site provides a basis for predicting the polarity of drug binding in the minor groove. Stereo drawings of the CC-1065-DNA adduct are proposed that are predictive of features of the CC-1065-DNA adduct elucidated in this investigation.     

Depletion of nicotinamide adenine dinucleotide in normal and xeroderma pigmentosum fibroblast cells by the antitumor drug CC-1065

CC-1065 is an extremely potent antitumor antibiotic that forms a well-defined adduct with DNA in which the molecule lies within the minor groove and is covalently attached through N3 of adenine. Addition of CC-1065 to human fibroblast cells produced a prolonged depletion of the nicotinamide adenine dinucleotide (NAD) pool even at extremely low drug concentrations (0.01 microgram/mL). The depletion of NAD by CC-1065 was blocked by 3-aminobenzamide, which is consistent with a NAD depletion mechanism involving poly-(ADP-ribose) synthesis in response to a repair-induced DNA strand breakage event. Significantly, similar extents of NAD depletion were also evident in xeroderma pigmentosum cells of complementation groups A and D following exposure to CC-1065. Since this NAD depletion is presumably associated with repair-induced incision, the repair of CC-1065-DNA adducts can probably take place by a pathway distinct from that involved in repair of more conventional bulky DNA adducts. The prolonged depletion of NAD, even at low doses of drug, suggests that CC-1065 causes DNA damage that results in a delay or block in DNA excision repair between the excision and ligation steps.     

Recognition of the DNA helix stabilizing anthramycin-N2 guanine adduct by UVRABC nuclease

The binding of the anti-tumor antibiotic anthramycin to a defined linear DNA fragment was investigated using both exonuclease III and lambda exonuclease. We show that most of the guanine residues are reactive toward anthramycin; however, several guanine residues showed preferential reactivity for the drug. Using purified UVRA, UVRB and UVRC proteins we present evidence that these three proteins in concert are able to recognize and produce specific strand cleavage flanking anthramycin-DNA adducts. The cleavage of anthramycin adducts by UVRABC nuclease is specific and results in strand breaks at five or six bases 5' and three or four bases 3'-flanking an adduct. At some guanine residues single incisions were observed only on one side of the adduct. The 5' strand breaks observed often occurred as doublet bands on sequencing gels, indicating plasticity in the site of 5' cleavage whereas the 3' cleavage did not show this effect. When DNA fragments modified with elevated levels of anthramycin were used as substrates the activity of the UVRABC nuclease toward the anthramycin adducts decreased. Possible mechanisms for the recognition and specific cleavage of the helix-stabilizing anthramycin DNA adduct and other helix destabilizing lesions by the UVRABC nuclease are discussed.     

Repair of helix-stabilizing anthramycin-N2 guanine DNA adducts by UVRA and UVRB proteins.

The transfectivity of anthramycin (Atm)-modified phi X174 replicative form (RF) DNA in Escherichia coli is lower in uvrA and uvrB mutant cells but much higher in uvrC mutant cells compared to wild-type cells. Pretreatment of the Atm-modified phage DNA with purified UVRA and UVRB significantly increases the transfectivity of the DNA in uvrA or uvrB mutant cells. This pretreatment greatly reduces the UVRABC nuclease-sensitive sites (UNSS) and Atm-induced absorbance at 343 nm in the Atm-modified DNA without producing apurinic sites. The reduction of UNSS is proportional to the concentrations of UVRA and UVRB and the enzyme-DNA incubation time and requires ATP. We conclude that there are two different mechanisms for repairing Atm-N2 guanine adducts by UVR proteins: (1) UVRA and UVRB bind to the Atm-N2 guanine double-stranded DNA region and consequently release the Atm from the adducted guanine; (2) UVRABC makes an incision at both sides of the Atm-DNA adduct. The latter mechanism produces potentially lethal double-strand DNA breaks in Atm-modified phi X174 RF DNA in vitro.     

Excision repair of adozelesin-N3 adenine adduct by 3-methyladenine-DNA glycosylases and UvrABC nuclease

Adozelesin is a synthetic analog of the antitumor antibiotic CC-1065, which alkylates the N3 of adenine in the minor groove in a sequence-selective manner. Since the cytotoxic potency of a DNA alkylating agent can be modulated by DNA excision repair system, we investigated whether nucleotide excision repair (NER) and base excision repair (BER) enzymes are able to excise the bulky DNA adduct induced by adozelesin. The UvrABC nuclease and 3-methyladenine-DNA glycosylase, that exhibit a broad spectrum of substrate specificity, were selected as typical NER and BER enzymes, respectively. The adozelesin-DNA adduct was first formed in the radiolabeled restriction DNA fragment and its excision by purified repair enzymes was monitored on a DNA sequencing gel. The treatment of the DNA adduct with a purified UvrABC nuclease and sequencing gel analysis of cleaved DNA showed that UvrABC nuclease was able to incise the adozelesin adduct. The incision site corresponded to the general nuclease incision site. Excision of this adduct by 3-methyladenine-DNA glycosylases was determined following the treatment of the DNA adduct with a homogeneous recombinant bacterial, rat and human 3-methyladenine-DNA glycosylases. Abasic sites generated by DNA glycosyalses were cleaved by the associated lyase activity of the E. coli formamidopyrimidine-DNA glycosylase (Fpg). Resolution of cleaved DNA on a sequencing gel showed that the DNA glycosylase from different sources could not release the N3-adenine adducts. A cytotoxicity assay using E. coli repair mutant strains showed that E. coli mutant strains defective in the uvrA gene were more sensitive to cell killing by adozelesin than E. coli mutant strain defective in the alkA gene or the wild type. These results suggest that the NER pathway seems to be the major excision repair system in protecting cells from the cytotoxicity of adozelesin.     

Quantification of aminofluorene adduct formation and repair in defined DNA sequences in mammalian cells using the UVRABC nuclease

Using the UVRABC nuclease as a reagent coupled with DNA restriction and hybridization analysis we have developed a method to quantify N-acetoxy-2-acetylaminofluorene (NAAAF)-induced DNA damage in the coding and noncoding sequences of the dihydrofolate reductase (DHFR) gene in Chinese hamster ovary (CHO) cells. High performance liquid chromatography analysis shows that the only DNA adduct formed in NAAAF-treated CHO cells is N-(deoxyguanosine-C8-yl)-2-aminofluorene (dG-C8-AF). DNA sequencing analysis demonstrates that the UVRABC nuclease incises at all potential sites in which dG-C8-AF adduct may form in linear DNA fragments. We have found that the formation and removal of dG-C8-AF adducts in the coding and 3' downstream noncoding sequences of the DHFR domain are similar in cells treated with 10 microM NAAAF (3.1 adducts/14 kilobases); DNA adduct removal attains 70% for both sequences within 24 h. This result contrasts with that obtained for the repair of cyclobutane dipyrimidines in the DHFR gene, in which the repair efficiency is much higher in the coding region than in the 3' downstream noncoding region. Our results suggest that in CHO cells the repair pathway for aminofluorene DNA adducts is not the same as that for cyclobutane dipyrimidines. This new technique has the potential to detect a variety of chemical carcinogen induced DNA adducts at the gene level in cultured cells and in DNA isolated from animal tissues.     

ABC excinuclease incises both 5' and 3' to the CC-1065-DNA adduct and its incision activity is stimulated by DNA helicase II and DNA polymerase I

CC-1065 is a large molecule that binds covalently to adenine residues of DNA in a sequence-specific manner and lies in the minor groove about four bases to the 5' side of the adducted residue. Using a reconstituted Escherichia coli nucleotide excision repair system, we have obtained data showing that the ABC excinuclease makes incisions both 5' and 3' to the CC-1065 adduct and that the incision activity is stimulated by the addition of helicase II and DNA polymerase I (and dNTPs). Our results with the CC-1065 adduct are consistent with the reported in vitro processing of other adducts (e.g., cisplatin, UV photoproducts) but do not agree with a recent study that reported anomalous processing of the CC-1065 adduct by ABC excinuclease and helicase II. Our results also imply that, in binding to damaged DNA, ABC excinuclease does not make important contacts in the minor groove four bases to the 5' side of the damaged residue.     

An NMR study of the covalent and noncovalent interactions of CC-1065 and DNA

The binding of the antitumor drug CC-1065 has been studied with nuclear magnetic resonance (NMR) spectroscopy. This study involves two parts, the elucidation of the covalent binding site of the drug to DNA and a detailed investigation of the noncovalent interactions of CC-1065 with a DNA fragment through analysis of 2D NOE (NOESY) experiments. A CC-1065-DNA adduct was prepared, and an adenine adduct was released upon heating. NMR (1H and 13C) analysis of the adduct shows that the drug binds to N3 of adenine by reaction of its cyclopropyl group. The reaction pathway and product formed were determined by analysis of the 13C DEPT spectra. An octamer duplex, d(CGATTAGC.GCTAATCG), was synthesized and used in the interaction study of CC-1065 and the oligomer. The duplex and the drug-octamer complex were both analyzed by 2D spectroscopy (COSY, NOESY). The relative intensity of the NOEs observed between the drug (CC-1065) and the octamer duplex shows conclusively that the drug is located in the minor groove, covalently attached to N3 of adenine 6 and positioned from the 3'----5' end in relation to strand A [d(CGATTA6GC)]. A mechanism for drug binding and stabilization can be inferred from the NOE data and model-building studies.     

Studies on the role of the phi X174 gene A protein in phi X viral strand synthesis. I. Replication of DNA containing an alteration in position 1 of the 30-nucleotide icosahedral bacteriophage origin

The influence of a C----G transversion at position 1 of the 30-base pair replication origin of bacteriophage phi X174 replicative form I DNA (phi X RFI) was examined in the RF----single-stranded circular DNA replication pathway catalyzed by the combined action of the purified phi X A protein, the Escherichia coli DNA polymerase III holoenzyme, rep helicase, and single-stranded DNA binding protein (Eisenberg, S., Scott, J.F., and Kornberg, A. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 1594-1597; Reinberg, D., Zipursky, S.L., and Hurwitz, J. (1981) J. Biol. Chem. 256, 13143-13151). RFI DNA containing this transversion was cleaved to RFII by the phi X A protein as effectively as DNA containing the wild-type origin. The altered duplex DNA, however, supported replication at a slower rate (3- to 4-fold) than the wild-type DNA due to a defect in the termination and reinitiation reactions catalyzed by the phi X A protein. This defect resulted in the accumulation of DNA products containing long single strands covalently joined to the mutant DNA. These single strands were susceptible to nuclease S1 and exonuclease VII attack. The defect in the template DNA containing C----G transversion was not corrected when this mutant origin was placed on the same strand with a wild-type origin. This double-origin DNA was also replicated poorly and led to the accumulation of large products, in contrast to the products formed with RFI DNA containing two wild-type 30-base pair replication origins on the same strand.     

DNA sequence recognition by the antitumor antibiotic CC-1065 and analogs of CC-1065

The DNA base pair preferences of the antitumor antibiotic CC-1065 and two analogs of CC-1065 were studied by following the rate of covalent bond formation (N-3 adenine adduct) with DNA oligomers containing the 5'NNTTA* and 5'NNAAA* sequences (N = nucleotide, A* = alkylated adenine). The rate of adduct formation of CC-1065 is greatly affected by DNA base changes at the fourth and fifth positions of the bonding site for the 5'NNAAA sequences, but not the 5'NNTTA sequences. However, an analog of CC-1065 containing the same alkylating moiety as CC-1065, but not the third fused ring system or additional methylene and oxygen substituents, shows similar rates of adduct formation for all sequences. A second analog of CC-1065 containing three fused ring systems, but not the methylene and oxygen substituents of CC-1065, shows rates of adduct formation with the same sequence dependence as CC-1065, but does not distinguish between the sequences to the degree shown by CC-1065. Adduct formation of CC-1065, but not the analogs, competes with a reversibly bound species. Thymine bases to the 3' side of a potentially reactive adenine or a cytosine base at the fifth position from the bonding adenine create reversible binding sites which decrease the rate of adduct formation of CC-1065. The sequence 5'GCGAATT binds CC-1065 only reversibly. This sequence can compete for CC-1065 with covalent bonding sequences if the sites are located in different oligomers, or if the sites are located (overlapped or not overlapped) in the same oligomer. The results of these competitive binding experiments suggest that the transfer of CC-1065 from the reversible binding site to the covalent bonding site with both sites located on a single DNA duplex, not overlapped, occurs through an equilibrium of CC-1065 in solution, not by migration of CC-1065 in the minor groove.     

Studies of the recognition sequence of phi X174 gene A protein. Cleavage site of phi X gene A protein in St-1 RFI DNA.

It is already known that phi X gene A protein converts besides phi X RFI DNA also the RFI DNAs of the single-stranded bacteriophages G4, St-1, alpha 3 and phi K into RFII DNA. We have extended this observations for bacteriophages G14 and U3. Restriction enzyme analysis placed the phi X gene A protein cleavage site in St-1 RF DNA in the HinfI restriction DNA fragment F10 and in the overlapping HaeIII restriction DNA fragment Z7. The exact position and the nucleotide sequence at the 3'-OH end of the nick were determined by DNA sequence analysis of the single-stranded DNA subfragment of the nicked DNA fragment F10 obtained by gelelectrophoresis in denaturing conditions. A stretch of 85 nucleotides of St-1 DNA around the position of the phi X gene A protein cleavage site was established by DNA sequence analysis of the restriction DNA fragment Z7F1. Comparison of this nucleotide sequence with the previously determined nucleotide sequence around the cleavage site of phi X gene A protein in phi X174 RF DNA and G4 RF DNA revealed an identical sequence of only 10 nucleotides. The results suggest that the recognition sequence of the phi X174 gene A protein lies within these 10 nucleotides.     

Calf thymus DNA binding/bonding properties of CC-1065 and analogs as related to their biological activities and toxicities.

CC-1065 is a potent natural antitumor antibiotic that binds non-covalently and covalently (N-3 adenine adduct) in the minor groove of B-form DNA. Synthetic analogs of CC-1065 do not exhibit the delayed death toxicity of CC-1065 and are efficacious anticancer agents, some of them curative in murine tumor models. In an attempt to understand the different biological properties of CC-1065 and analogs, we have determined the following quantities for CC-1065, enantiomeric CC-1065, and three biologically active analogs and their enantiomers: the calf thymus DNA (CT-DNA) induced molar ellipticity of the adduct (or how rigidly the adduct is held in the right-hand conformation of the minor groove); the stability of the adduct with respect to long incubation times and to digestion by snake venom phosphodiesterase I (SVPD); the stabilizing effect on the CT-DNA helix of the covalently and non-covalently bound species with respect to thermal melting; and the CT-DNA binding/bonding (non-covalent/covalent) profiles at a low molar ratio of nucleotide to drug. The major observations from these studies are as follows: (i) molecules which show large DNA interaction parameters, stable adducts, and significant non-covalent binding exhibit delayed death toxicity; (ii) molecules which show intermediate DNA interaction parameters and stable adducts, but do not show significant non-covalent binding, do not exhibit delayed death toxicity and are biologically active; (iii) molecules which show small DNA interaction parameters and unstable DNA adducts are biologically inactive. The results suggest that a window exists in the affinity for the minor groove of DNA wherein an analog may possess the correct balance of toxicity and activity to make a useful anticancer agent. Outside of this window, the analog causes delayed deaths or has no significant biological activity.     

Circular and linear simian virus 40 DNAs differ in recombination.

Linear forms of simian virus 40 (SV40) DNA, when added to transfection mixtures containing circular SV40 and phi X174 RFI DNAs, enhanced the frequency of SV40/phi X174 recombination, as measured by infectious center in situ plaque hybridization in monkey BSC-1 cells. The sequences required for the enhancement of recombination by linear DNA reside within the SV40 replication origin/regulatory region (nucleotides 5,171 to 5,243/0 to 128). Linearization of phi X174 RFI DNA did not increase the recombination frequency. The SV40/phi X174 recombinant structures arising from transfections supplemented with linear forms of origin-containing SV40 DNA contained phi X174 DNA sequences interspersed within tandem head-to-tail repeats derived from the recombination-enhancing linear DNA. Evidence is presented that the tandem repeats are not formed by homologous recombination and that linear forms of SV40 DNA must compete with circular SV40 DNA for the available T antigen to enhance recombination. We propose that the enhancement of recombination by linear SV40 DNA results from the entry of that DNA into a rolling circle type of replication pathway which generates highly recombinogenic intermediates.     

DNA strand breakage by hydroxyphenyl radicals generated from mutagenic diazoquinone compounds.

The mutagenic diazoquinone compounds p-diazoquinone (p-DQ), o-diazoquinone (o-DQ) and 3-diazo-N-nitrosobamethan (D-BM) cleaved the phosphodiester bond of lambda DNA, phi X174 RFI DNA and M13mp8ss DNA. p-DQ also cleaved the phosphodiester bond of bis(p-nitrophenyl)phosphate. The breakage of the phosphodiester bond was inhibited by the antioxidant butyl hydroxyanisole (BHA), ethanol, the spin trapping agent DMPO, cysteine and 2-mercaptoethanol. While incubation of p-DQ and o-DQ alone gave p-hydroquinone and catechol, respectively, incubation of these compounds in the presence of BHA and ethanol gave phenol in large yields. Incubation of p-DQ and o-DQ with the spin trapping agents DMPO and PBN gave spin adducts assignable as p- and o-hydroxyphenyl adducts, respectively. The breakage of the phosphodiester bond of DNA by the diazoquinone compounds is suggested to be due to the hydroxyphenyl radicals generated during incubation.     

Characterization of an adduct between CC-1065 and a defined oligodeoxynucleotide duplex.

CC-1065 is a potent antitumor antibiotic produced by Streptomyces zelensis. The drug binds covalently through N-3 of adenine and lies within the minor groove of DNA. Previous studies indicated that CC-1065 reacted with adenine in DNA to yield a thermally labile product that could be used to reveal its sequence specificity. These studies also provided insight into a DNA sequence (5'-CGGAGTTAGGGGCG-3') which should bind one molecule of CC-1065 in an unambiguous manner. This sequence, which contains the CC-1065 adenine binding site within the sequence 5'-TTA-3' was chemically synthesized together with the complementary strand. CC-1065 reacted with the oligoduplex to give an adduct that maintained the B-DNA form and had a final CD spectrum similar to those of the CC-1065 complexes formed with calf thymus DNA. The above 14mer was 5' end-labelled with 32P, annealed with its complementary strand, reacted with CC-1065 and heated. Drug-mediated strand breakage was evaluated on a sequencing gel. A single break occurred in the labelled strands to give a fragment that migrated as an 8.5mer; subsequent piperidine treatment produced a fragment that migrated as a 7mer, which is the size expected from the known binding of CC-1065 at adenine in 5'-TTA-3' sequences.     

Reversibility of the covalent reaction of CC-1065 and analogues with DNA.

Covalent DNA adducts of the antitumor antibiotic CC-1065 and its analogues undergo a retrohomologous Michael reaction in aqueous/organic solvent mixtures to regenerate the initial cyclopropylpyrroloindole (CPI) structure and, presumably, intact DNA. This reaction, which at higher temperatures competes with depurination of the N3-alkylated adenine, also occurs to a significant extent at 37 degrees C in neutral aqueous solution. Tritium-labeled adozelesin, covalently bonded to a 3-kilobase DNA restriction fragment which was exhaustively extracted to remove unbonded drug, was efficiently transferred to a 1-kilobase fragment upon coincubation for 20 h at 37 degrees C in aqueous buffer. Covalent adducts of adozelesin, but not CC-1065, on calf thymus DNA were cytotoxic to L1210 cells after incubation for 3 days at 37 degrees C, indicating that reversal of DNA alkylation can mediate potent cellular effects for simplified CC-1065 analogues.