Recognition and incision of oxidative intrastrand cross-link lesions by UvrABC nuclease

Nucleotide excision repair (NER) is a repair pathway that removes a variety of bulky DNA lesions in both prokaryotic and eukaryotic cells. The perturbation of DNA helix structure caused by the oxidative intrastrand lesions could render them good substrates for the NER pathway. Here we employed Escherichia coli NER enzymes, i.e., UvrA, UvrB, and UvrC, to examine the incision efficiency of duplex DNA carrying three different oxidative intrastrand cross-link lesions, that is, G[8-5]C, G[8-5m]mC, and G[8-5m]T, and two dithymine photoproducts, namely, the cis,syn-cyclobutane pyrimidine dimer (T[c,s]T) and the pyrimidine(6-4)pyrimidone product (T[6-4]T). Our results showed that T[6-4]T was the best substrate for UvrA binding, followed by G[8-5]C, G[8-5m]mC, and G[8-5m]T, and then by T[c,s]T. The efficiencies of the UvrABC incisions of these lesions were consistent with their UvrA binding affinities: the stronger the binding to UvrA, the higher the rate of incision. In addition, flanking DNA sequences appeared to have little effect on the binding affinity of UvrA for G[8-5]C as AG[8-5]CA was only slightly preferred over CG[8-5]CG. Consistently, these two sequences exhibited almost no difference in incision rates. Furthermore, we investigated the thermal stability of dodecameric duplexes containing G[8-5m]mC or G[8-5m]T, and our results revealed that these two lesions destabilized the duplex, due to an increase in the free energy for duplex formation at 37 degrees C, by approximately 5.4 and 3.6 kcal/mol, respectively. The destabilizations to the DNA helix caused by those lesions, for the most part, are correlated with the binding affinities of UvrA and incision rates of UvrABC. Taken together, the results from this study suggest that oxidative intrastrand lesions might be substrates for NER enzymes in vivo.     

Comparative direct-acting mutagenicity of 1- and 2-nitropyrene: Evidence for 2-nitropyrene mutagenesis by both guanine and adenine adducts

The mutations and DNA adducts produced by the environmental pollutant 2-nitropyrene were examined in Salmonella typhimurium tester strains. 2-Nitropyrene was a stronger mutagen than its extensively studied structural isomer 1-nitropyrene in strains TA96, TA97, TA98, TA100, TA102, TA104 and TA1538. Both 1- and 2-nitropyrene were essentially inactive in TA1535. The mutagenicity of 1- and 2-nitropyrene in TA98 was much higher than in TA98NR and the activity of these compounds in TA100 was much higher than in TA100NR. While 1-nitropyrene exhibited similar mutagenicity in strains TA98 and TA98/1,8-DNP₆, the mutagenicity of 2-nitropyrene in TA98/1,8-DNP₆ was much lower than in TA98. Analysis of DNA from TA96 and TA104 incubated with 2-nitropyrene indicated the presence of two adducts, N-(deoxyguanosin-8-yl)-2-aminopyrene and N-deoxyadenosin-8-yl)-2-aminopyrene. The results suggest that 2-nitropyrene is metabolized by bacterial nitroreductase(s) to N-hydroxy-2-aminopyrene, and possibly by activation to a highly mutagenic O-acetoxy ester. DNA adduct formation with deoxyguanosine and deoxyadenosine correlates with the mutagenicity of 2-nitropyrene in tester strains possessing both G:C and A:T mutational targets.     

Differential incision of bulky carcinogen-DNA adducts by the UvrABC nuclease: comparison of incision rates and the interactions of Uvr subunits with lesions of different structures

The UvrABC nuclease system from Escherichia coli removes DNA damages induced by a wide range of chemical carcinogens with variable efficiencies. The interactions with UvrABC proteins of the following three lesions site-specifically positioned in DNA, and of known conformations, were investigated: (i) adducts derived from the binding of the (-)-(7S,8R,9R,10S) enantiomer of 7,8-dihydroxy-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(-)-anti-BPDE] by cis-covalent addition to N(2)-2'-deoxyguanosine [(-)-cis-anti-BP-N(2)-dG], (ii) an adduct derived from the binding of the (+)-(1R,2S,3S,4R) enantiomer of 1,2-dihydroxy-3,4-epoxy-1,2,3, 4-tetrahydro-5-methylchrysene [(+)-anti-5-MeCDE] by trans addition to N(2)-2'-deoxyguanosine [(+)-trans-anti-MC-N(2)-dG], and (iii) a C8-2'-deoxyguanosine adduct (C8-AP-dG) formed by reductively activated 1-nitropyrene (1-NP). The influence of these three different adducts on UvrA binding affinities, formation of UvrB-DNA complexes by quantitative gel mobility shift analyses, and the rates of UvrABC incision were investigated. The binding affinities of UvrA varied among the three adducts. UvrA bound to the DNA adduct (+)-trans-anti-MC-N(2)-dG with the highest affinity (K(d) = 17 +/- 2 nM) and to the DNA containing C8-AP-dG with the least affinity (K(d) = 28 +/- 1 nM). The extent of complex formation with UvrB was also the lowest with the C8-AP-dG adduct. 5' Incisions occurred at the eighth phosphate from the modified guanine. The major 3' incision site corresponded to the fifth phosphodiester bond for all three adducts. However, additional 3' incisions were observed at the fourth and sixth phosphates in the case of the C8-AP-dG adduct, whereas in the case of the (-)-cis-anti-BP-N(2)-dG and (+)-trans-anti-MC-N(2)-dG lesions additional 3' cleavage occurred at the sixth and seventh phosphodiester bonds. Both the initial rate and the extent of 5' and 3' incisions revealed that C8-AP-dG was repaired less efficiently in comparison to the (-)-cis-anti-BP-N(2)-dG and (+)-trans-anti-MC-N(2)-dG containing DNA adducts. Our study showed that UvrA recognizes conformational changes induced by structurally different lesions and that in certain cases the binding affinities of UvrA and UvrB can be correlated with the incision rates. The size of the bubble formed around the damaged site with mismatched bases also appears to influence the incision rates. A particularly noteworthy finding in this study is that UvrABC repair of a substrate with no base opposite C8-AP-dG was quite inefficient as compared to the same adduct with a C opposite it. These findings are discussed in terms of the available NMR solution structures.     

Differential effects of N-acetyl-2-aminofluorene and N-2-aminofluorene adducts on the conformational change in the structure of DNA polymerase I (Klenow fragment)

The carcinogen N-acetyl-2-aminofluorene forms two major DNA adducts: the N-(2'-deoxyguanosin-8-yl)-2-acetylaminofluorene adduct (dG-C8-AAF) and its deacetylated derivative, the N-(2'-deoxyguanosin-8-yl)-2-aminofluorene adduct (dG-C8-AF). It is well established that the AAF adduct is a very strong block for DNA synthesis in vitro while the AF adduct is more easily bypassed. In an effort to understand the molecular mechanism of this phenomenon, the structure of the complex of an exonuclease-deficient Escherichia coli DNA polymerase I (Klenow fragment) bound to primer-templates containing either an AF or AAF adduct in or near the active site was probed by nuclease and protease digestion analyses. The results of these experiments suggest that positioning the AAF adduct in the polymerase active site strongly inhibits the conformational change that is required for the insertion of a nucleotide. Similar experiments with AF-modified primer-templates shows a much less pronounced effect. The inhibition of the conformational change by either adduct is not detected if they are positioned in the single-stranded part of the template just one nucleotide before the active site. These findings may explain the different abilities of these lesions to block DNA synthesis.     

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.     

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.     

Effects of DNA adduct structure and sequence context on strand opening of repair intermediates and incision by UvrABC nuclease

DNA damage recognition of nucleotide excision repair (NER) in Escherichia coli is achieved by at least two steps. In the first step, a helical distortion is recognized, which leads to a strand opening at the lesion site. The second step involves the recognition of the type of chemical modification in the single-stranded region of DNA during the processing of the lesions by UvrABC. In the current work, by comparing the efficiencies of UvrABC incision of several types of different DNA adducts, we show that the size and position of the strand opening are dependent on the type of DNA adducts. Optimal incision efficiency for the C8-guanine adducts of 2-aminofluorene (AF) and N-acetyl-2-aminofluorene (AAF) was observed in a bubble of three mismatched nucleotides, whereas the same for C8-guanine adduct of 1-nitropyrene and N(2)-guanine adducts of benzo[a]pyrene diol epoxide (BPDE) was noted in a bubble of six mismatched nucleotides. This suggests that the size of the aromatic ring system of the adduct might influence the extent and number of bases associated with the opened strand region catalyzed by UvrABC. We also showed that the incision efficiency of the AF or AAF adduct was affected by the neighboring DNA sequence context, which, in turn, was the result of differential binding of UvrA to the substrates. The sequence context effect on both incision and binding disappeared when a bubble structure of three bases was introduced at the adduct site. We therefore propose that these effects relate to the initial step of damage recognition of DNA structural distortion. The structure-function relationships in the recognition of the DNA lesions, based on our results, have been discussed.     

Repair of mitomycin C mono- and interstrand cross-linked DNA adducts by UvrABC: a new model

Mitomycin C induces both MC-mono-dG and cross-linked dG-adducts in vivo. Interstrand cross-linked (ICL) dG-MC-dG-DNA adducts can prevent strand separation. In Escherichia coli cells, UvrABC repairs ICL lesions that cause DNA bending. The mechanisms and consequences of NER of ICL dG-MC-dG lesions that do not induce DNA bending remain unclear. Using DNA fragments containing a MC-mono-dG or an ICL dG-MC-dG adduct, we found (i) UvrABC incises only at the strand containing MC-mono-dG adducts; (ii) UvrABC makes three types of incisions on an ICL dG-MC-dG adduct: type 1, a single 5′ incision on 1 strand and a 3′ incision on the other; type 2, dual incisions on 1 strand and a single incision on the other; and type 3, dual incisions on both strands; and (iii) the cutting kinetics of type 3 is significantly faster than type 1 and type 2, and all of 3 types of cutting result in producing DSB. We found that UvrA, UvrA + UvrB and UvrA + UvrB + UvrC bind to MC-modified DNA specifically, and we did not detect any UvrB- and UvrB + UvrC–DNA complexes. Our findings challenge the current UvrABC incision model. We propose that DSBs resulted from NER of ICL dG-MC-dG adducts contribute to MC antitumor activity and mutations.     

Recognition and activation of Rho GTPases by Vav1 and Vav2 guanine nucleotide exchange factors

Vav proteins are Rho GTPase-specific guanine nucleotide exchange factors (GEFs) that are distinguished by the tandem arrangement of Dbl homology (DH), Pleckstrin homology (PH), and cysteine rich domains (CRD). Whereas the tandem DH-PH arrangement is conserved among Rho GEFs, the presence of the CRD is unique to Vav family members and is required for efficient nucleotide exchange. We provide evidence that Vav2-mediated nucleotide exchange of Rho GTPases follows the Theorell-Chance mechanism in which the Vav2.Rho GTPase complex is the major species during the exchange process and the Vav2.GDP-Mg(2+).Rho GTPase ternary complex is present only transiently. The GTPase specificity for the DH-PH-CRD Vav2 in vitro follows this order: Rac1 > Cdc42 > RhoA. Results obtained from fluorescence anisotropy and NMR chemical shift mapping experiments indicate that the isolated Vav1 CRD is capable of directly associating with Rac1, and residues K116 and S83 that are in the proximity of the P-loop and the guanine base either are part of this binding interface or undergo a conformational change in response to CRD binding. The NMR studies are supported by kinetic measurements on Rac1 mutants S83A, K116A, and K116Q and Vav2 CRD mutant K533A in that these mutants affect both the initial binding event of Vav2 with Rac1 (k(on)) and the rate-limiting dissociation of Vav2 from the Vav2.Rac1 binary complex (thereby influencing the enzyme turnover number, k(cat)). The results suggest that the CRD domain in Vav proteins plays an active role, affecting both the k(on) and the k(cat) for Vav-mediated nucleotide exchange on Rho GTPases.     

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.     

Ability of adducts formed in a shuttle vector by reactive metabolites of 1-nitropyrene and benzo(a)pyrene to induce mutations when the plasmid replicates in human cells

An SV40-based shuttle vector, pZ189, carrying a bacterial suppressor tRNA target gene (supF), was treated with radiolabeled polycyclic aromatic carcinogens and the number of covalently bound residues (adducts) per plasmid was determined. The plasmids were transfected into human cell line 293, allowed to replicate, and the progeny plasmids rescued and assayed for the frequency of supF mutants. The agents tested were the 7,8-diol-9,10-epoxide of benzo(a)pyrene (BPDE) and 1-nitrosopyrene (1-NOP). With each agent there was a linear increase in the frequency of supF mutants as a function of the number of DNA adducts formed, reaching frequencies 15 to 25 times higher than the background frequency of 1.4 x 10(-4). When compared on the basis of adducts formed per plasmid BPDE, which forms its principal DNA adduct at the N2 position of guanine, was approximately four times more mutagenic than 1-NOP, which binds principally at the C8 position of guanine. This difference in mutagenic effectiveness may reflect intrinsic differences in the nature of the adducts and their location in the DNA molecule, but it could also reflect a difference in the rate of removal of particular adducts by nucleotide excision repair since the 293 host cell line excised BPDE-induced adducts from genomic DNA at least three times slower than 1-NOP-induced adducts. Agarose gel electrophoresis and DNA sequencing analysis of mutants derived from untreated plasmids showed that the majority (70%) involved deletions, insertions, or altered gel mobility (gross rearrangements). In contrast, the majority of those derived from carcinogen-treated plasmids were base substitutions. DNA sequencing of 86 unequivocally independent mutants derived from BPDE-treated plasmid and 60 from 1-NOP-treated plasmid indicated that 70% to 80% contained a single base substitution, 5%-10% had two base substitutions, and 4%-10% had small insertions or deletions (one or two basepairs). The majority (83%) of the base substitutions in mutants from BPDE- or 1-NOP-treated plasmid were transversions, mainly G.C----T.A. Each carcinogen produced its own spectrum of mutations.     

Comparative mutagenesis of the C8-guanine adducts of 1-nitropyrene and 1,6- and 1,8-dinitropyrene in a CpG repeat sequence. A slipped frameshift intermediate model for dinucleotide deletion

In the Ames Salmonella typhimurium reversion assay 1,6- and 1,8-dinitropyrenes (1,6- and 1,8-DNPs) are much more potent mutagens than 1-nitropyrene (1-NP). Genetic experiments established that certain differences in the metabolism of the DNPs, which in turn result in increased DNA adduction, play a role. It remained unclear, however, if the DNP adducts, N-(guanin-8-yl)-1-amino-6 ()-nitropyrene (Gua-C8-1,6-ANP and Gua-C8-1,8-ANP), which contain a nitro group on the pyrene ring covalently linked to the guanine C8, are more mutagenic than the major 1-NP adduct, N-(guanin-8-yl)-1-aminopyrene (Gua-C8-AP). In order to address this, we have compared the mutation frequency of the three guanine C8 adducts, Gua-C8-AP, Gua-C8-1,6-ANP, and Gua-C8-1,8-ANP in a CGCG*CG sequence. Single-stranded M13mp7L2 vectors containing these adducts and a control were constructed and replicated in Escherichia coli. A remarkable difference in the induced CpG deletion frequency between these adducts was noted. In repair-competent cells the 1-NP adduct induced 1.7% CpG deletions without SOS, whereas the 1,6- and 1,8-DNP adducts induced 6.8 and 10.0% two-base deletions, respectively. With SOS, CpG deletions increased up to 1.9, 11.1, and 15.1% by 1-NP, 1,6-, and 1,8-DNP adducts, respectively. This result unequivocally established that DNP adducts are more mutagenic than the 1-NP adduct in the repetitive CpG sequence. In each case the mutation frequency was significantly increased in a mutS strain, which is impaired in methyl-directed mismatch repair, and a dnaQ strain, which carries a defect in proofreading activity of the DNA polymerase III. Modeling studies showed that the nitro group on the pyrene ring at the 8-position can provide additional stabilization to the two-nucleotide extrahelical loop in the promutagenic slipped frameshift intermediate through its added hydrogen-bonding capability. This could account for the increase in CpG deletions in the M13 vector with the nitro-containing adducts compared with the Gua-C8-AP adduct itself.     

Enhancement of the human antibody response by C8-substituted guanine ribonucleosides in synergy with interleukin 2

The antigen-specific primary antibody response of human lymphocytes in vitro was studied with respect to dependency upon the lymphokine interleukin 2 (IL 2) and its subsequent modulation by C8-substituted guanine ribonucleosides. The specific response to sheep erythrocytes was shown to be dependent on the presence of IL 2 in culture. However, addition of optimal concentrations of the nucleoside, 7-methyl-8-oxoguanosine (7m8oGuo), to cultures containing antigen and IL 2 resulted in marked amplification of the underlying antibody response. This synergistic effect between 7m8oGuo and IL 2 was antigen dependent and could not be accounted for by summation of the independent antigen-specific and nonspecific (polyclonal) components. That IL 2 itself was in fact responsible for both the specific response to antigen and the synergistic interaction with 7m8oGuo was confirmed in experiments with purified IL 2 produced by recombinant DNA technology. The response to antigen was enhanced by 7m8oGuo in a dose-dependent fashion. The results of kinetic studies demonstrated that this nucleoside is fully effective within the context of an ongoing immune response, because addition of 7m8oGuo could be delayed up to 3 days of the 6-day culture period without loss of subsequent immunoenhancement. Lymphocyte populations largely depleted of T cells were capable of mounting vigorous responses to antigen in the presence of 7m8oGuo so long as IL 2, either partially purified or purified recombinant material, was added to culture.     

Induction of double-strand breaks by S1 nuclease, mung bean nuclease and nuclease P1 in DNA containing abasic sites and nicks.

Defined DNA substrates containing discrete abasic sites or paired abasic sites set 1, 3, 5 and 7 bases apart on opposite strands were constructed to examine the reactivity of S1, mung bean and P1 nucleases towards abasic sites. None of the enzymes acted on the substrate containing discrete abasic sites. Under conditions where little or no non-specific DNA degradation was observed, all three nucleases were able to generate double-strand breaks when the bistranded abasic sites were 1 and 3 base pairs apart. However, when the abasic sites were further apart, the enzymes again failed to cleave the DNA. These results indicate that single abasic sites do not cause sufficient denaturation of the DNA to allow incision by these single-strand specific endonucleases. The reactivity of these enzymes was also investigated on DNA substrates that were nicked by DNasel or more site-specifically by endonuclease III incision at the discrete abasic sites. The three nucleases readily induced a strand break opposite such nicks.     

The N2-guanine adduct but not the C8-guanine or N6-adenine adducts formed by 4-nitroquinoline 1-oxide blocks the 3'-5' exonuclease action of T4 DNA polymerase

When O-acetyl-4-(hydroxyamino)quinoline 1-oxide (Ac-4HAQO) reacts with double-stranded DNA at 37 degrees C the major products, N2-guanine, C8-guanine, and N6-adenine adducts, are formed in the proportions of 5:3:2, respectively. When the reaction is carried out with single-stranded DNA at 0 degree C, the products are found in the ratio 1:7:2. Unique 174-bp DNA fragments were modified in these ways and used as substrates for the 3'-5' exonuclease activity of T4 DNA polymerase. The results obtained showed that the exonuclease is blocked by the N2-guanine adduct but not the other two adducts. Interpretation of the cleavage patterns suggested that the enzyme stopped 2 nucleotides before the N2-guanine adduct. The N2-guanine adduct lies in the minor groove of the DNA double helix, while the other two adducts are found in the major groove. Apparently, only the former hinders progression of the enzyme.     

DNA replication-blocking properties of adducts formed by aflatoxin B1-2,3-dichloride and aflatoxin B1-2,3-oxide

The carcinogen aflatoxin B1 (AFB1), upon activation to a hypothesized AFB1-2,3-oxide (AFB1-oxide), reacts with DNA guanines. Aflatoxin B1-2,3-dichloride (AFB1-Cl2) was originally synthesized as an electronic analog for the putative AFB1-oxide, which has never been isolated due to presumed reactivity. We have previously shown that AFB1-oxide reacts with base-paired DNA guanines in a sequence-specific manner, as revealed by an alkali-degradation analysis. On the basis of a replication-block analysis, we have shown that AFB1-Cl2 reacts with single-stranded DNA preferentially at inverted repeat sequences, which were suggested to be capable of forming intrastrand base-paired structures. Here, we present data to show the following. Both AFB1-oxide and AFB1-Cl2 react with guanines in double-stranded DNA to induce similar sequence-specific, alkali-labile sites. Reactivity with partial DNA duplexes as well as the use of single-strand specific chemical probes directly demonstrates that AFB1-Cl2, like AFB1-oxide, prefers base-paired guanines over non-base-paired guanines. DNA replication block patterns induced by AFB1-oxide are essentially similar to those induced by AFB1-Cl2. Unexpectedly, and unlike other tested DNA lesions, Mn2+ does not appear to affect the template blocking properties of the adduct formed by AFB1-Cl2 or AFB1-oxide. The sites for replication stoppage as well as the lack of a Mn2+ effect on adducted templates have implications for the mechanisms of mutagenesis by activated AFB1.     

Endogenous formation and significance of 1,N2-propanodeoxyguanosine adducts

The detection of 1,N2-propanodeoxyguanosine adducts in the DNA of rodent and human tissues as endogenous lesions has raised important questions regarding the source of their formation and their roles in carcinogenesis. Both in vitro and in vivo studies have generated substantial evidence which supports the involvement of short- and long-chain enals derived from oxidized polyunsaturated fatty acids (PUFAs) in their formation. These studies show that: (1) the cyclic propano adducts are common products from reactions of enals with DNA bases; (2) they are formed specifically from linoleic acid (LA; omega-6) and docosahexaenoic acid (omega-3) under in vitro stimulated lipid peroxidation conditions; (3) the levels of propano adducts are dramatically increased in rat liver DNA upon depletion of glutathione; (4) the adduct levels are increased in the liver DNA of the CCl4-treated rats and the mutant strain of Long Evans rats which are genetically predisposed to increased lipid peroxidation; and (5) adduct levels are significantly higher in older rats than in newborn rats. These studies collectively demonstrate that tissue lipid peroxidation is a main endogenous pathway leading to propano adduction in DNA. The possible contribution from environmental sources, however, cannot be completely excluded. The mutagenicity of enals and the mutations observed in site-specific mutagenesis studies using a model 1,N2-propanodeoxyguanosine adduct suggest that these adducts are potential promutagenic lesions. The increased levels of the propano adducts in the tissue of carcinogen-treated animals also provide suggestive evidence for their roles in carcinogenesis. The involvement of these adducts in tumor promotion is speculated on the basis that oxidative condition in tissues is believed to be associated with this process.     

Quantitation of the 32P-postlabeling reaction using cyclic N1,N2 and C8 modified deoxyguanosine 3'-monophosphates as substrates.

The 32P-postlabeling technique was used to investigate the efficiency of phosphorylation reaction by T4 polynucleotide kinase using seven synthetic adducted deoxyguanosine 3'-monophosphates. The adducts included cyclic N1,N2 derivatives and the C8 adduct of 4-aminobiphenyl. The adducted substrates were detected at a subfemtomole sensitivity except for one of the diastereomeric propanoguanine derivatives. In general, the recommended conditions were found to be proper for an efficient phosphorylation of the adducts studied. Sensitivity of the adducts to the 3'-dephosphorylation reaction of nuclease P1 was also tested. All the complex cyclic adducts were resistant towards P1. However, the ethenoguanine and 4-aminobiphenyl adducts were relatively sensitive towards P1. No differences were noted between diastereomers.