Mutagenicity of the 1-nitropyrene-DNA adduct N-(deoxyguanosin-8-yl)-1-aminopyrene in mammalian cells.

The mutagenesis of the major DNA adduct N-(deoxyguanosin-8-yl)-1-aminopyrene (C8-AP-dG) formed by 1-nitropyrene was compared with the analogous C8-dG adducts of 2-aminofluorene (AF) and N-acetyl-2-aminofluorene (AAF) in simian kidney (COS-7) cells. The DNA sequence chosen for this comparison contained 5'-CCATC GCTACC-3' that has been used for solution NMR investigations. The structural and conformational differences among these lesions are well-established [Patel, D. J., Mao, B., Gu, Z., Hingerty, B. E., Gorin, A., Basu, A. K., and Broyde,S. (1998) NMR solution structures of covalent aromatic amine-DNA adducts and their mutagenic relevance. Chem. Res. Toxicol. 11, 391- 407.]. Accordingly, we found a notable difference in the viability of the progeny, which showed that the AAF adduct was most toxic and that the AF adduct was least toxic, with the AP adduct exhibiting intermediate toxicity. However, analysis of the progeny showed that translesion synthesis was predominantly error-free. Only low-level mutations (<3%) were detected with G-->T as the dominant type of mutation by all three DNA adducts. When C8-AP-dG was evaluated in a repetitive 5'-CGC GCG-3' sequence, higher mutational frequency ( approximately 8%) was observed. Again, G-->T was the major type of mutations in simian kidney cells, even though in bacteria CpG deletions predominate in this sequence [Hilario, P., Yan, S., Hingerty, B. E., Broyde, S., and Basu, A. K. (2002) 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. J. Biol. Chem. 277, 45068- 45074.]. Mutagenesis of C8-AP-dG in a 12-mer containing the local DNA sequence around codon 273 of the p53 tumor suppressor gene, where the adduct was located at the second base of this codon, was also investigated. In this 5'-GTGC GTGTTTGT-3' site, the mutations were slightly lower but not very different from the progeny derived from the 5'-CGC GCG-3' sequence. However, the mutational frequency increased by more than 50% when the 5'-C to the adduct was replaced with a 5-methylcytosine (5-MeC). With a 5-MeC, the most notable change in mutation was the enhancement of G-->A, which occurred 2.5 times relative to a 5'-C. The C8-AP-dG adduct in codon 273 dodecamer sequence with a 5'-C or 5-MeC was also evaluated in human embryonic kidney (293T) cells. Similar to COS cells, targeted mutations doubled with a 5-MeC 5' to the adduct. Except for an increase in G-->C transversions, the results in 293T were similar to that in COS cells. We conclude that C8-AP-dG mutagenesis depends on the type of cell in which it is replicated, the neighboring DNA sequence, and the methylation status of the 5'-C.

DNA adduct of the mitomycin C metabolite 2,7-diaminomitosene is a nontoxic and nonmutagenic DNA lesion in vitro and in vivo.

Mitomycin C (MC) is a cytotoxic and mutagenic antitumor agent that alkylates and cross-links DNA. These effects are dependent on reductive bioactivation of MC. 2,7-Diaminomitosene (2,7-DAM) is the major metabolite of MC in tumor cells, generated by the reduction of MC. 2,7-DAM alkylates DNA in the cell in situ, forming an adduct at the N7 position of 2'-deoxyguanosine (2,7-DAM-dG-N7). To determine the biological effects of this adduct, we have synthesized an oligonucleotide containing a single 2,7-DAM-dG-N7 adduct and inserted it into an M13 bacteriophage genome. Replication of this construct in repair-competent Escherichia coli showed that the adduct was only weakly toxic and generated approximately 50% progeny as compared to control. No mutant was isolated after analysis of more than 4000 progeny phages from SOS-induced or uninduced host cells; therefore, we estimate that the mutation frequency of 2,7-DAM-dG-N7 was less than 2 x 10(-4) in E. coli. Subsequently, to determine if this adduct might be mutagenic in mammalian cells, it was incorporated into a single-stranded shuttle phagemid vector, pMS2, and replicated in simian kidney (COS-7) cells. Analysis of the progeny showed that mutational frequency of a site specific 2,7-DAM-dG-N7 was not higher than the spontaneous mutation frequency in simian kidney cells. In parallel experiments in cell free systems, template oligonucleotides containing a single 2,7-DAM-dG-N7 adduct directed selective incorporation of cytosine in the 5'-32P-labeled primer strands opposite the adducted guanine, catalyzed by Klenow (exo-) DNA polymerase. The adducted templates also supported full extension of primer strands by Klenow (exo-) and T7 (exo-) DNA polymerases and partial extension by DNA polymerase eta. The innocuous behavior of the 2,7-DAM-dG-N7 monoadduct in vivo and in vitro is in sharp contrast to that of the toxic MC-dG-N2 monoadduct reported earlier.

Interactions of human replication protein A with single-stranded DNA adducts.

Human RPA (replication protein A), a single-stranded DNA-binding protein, is required for many cellular pathways including DNA repair, recombination and replication. However, the role of RPA in nucleotide excision repair remains elusive. In the present study, we have systematically examined the binding of RPA to a battery of well-defined ssDNA (single-stranded DNA) substrates using fluorescence spectroscopy. These substrates contain adducts of (6-4) photoproducts, N-acetyl-2-aminofluorene-, 1-aminopyrene-, BPDE (benzo[a]pyrene diol epoxide)- and fluorescein that are different in many aspects such as molecular structure and size, DNA disruption mode (e.g. base stacking or non-stacking), as well as chemical properties. Our results showed that RPA has a lower binding affinity for damaged ssDNA than for non-damaged ssDNA and that the affinity of RPA for damaged ssDNA depends on the type of adduct. Interestingly, the bulkier lesions have a greater effect. With a fluorescent base-stacking bulky adduct, (+)-cis-anti-BPDE-dG, we demonstrated that, on binding of RPA, the fluorescence of BPDE-ssDNA was significantly enhanced by up to 8-9-fold. This indicated that the stacking between the BPDE adduct and its neighbouring ssDNA bases had been disrupted and there was a lack of substantial direct contacts between the protein residues and the lesion itself. For RPA interaction with short damaged ssDNA, we propose that, on RPA binding, the modified base of ssDNA is looped out from the surface of the protein, permitting proper contacts of RPA with the remaining unmodified bases.

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.