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′-CCATCGCTACC-3′ that has been used for solution NMR investigations. The structural and conformational differences among these lesions are wellestablished (Patel et al., 1998). Accordingly, we found a notable difference in the viability of the progeny, which showed that the AAF adduct was most toxic and 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′-CGCGCG-3′ sequence, higher mutational frequency (~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 et al, 2002). 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′-GTGCGTGTTTGT-3′ site, the mutations were slightly lower but not very different from the progeny derived from the 5′-CGCGCG-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 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.Nucleotide excision repair (NER), 1 as one of the primary DNA repair pathways in cells, is capable of removing an extensive variety of bulky lesions, induced by chemicals and radiation, with varying efficiencies (1-3). Its ability to recognize and excise such a broad repertoire of substrates has been the subject of intensive research, and it is generally believed that the success of NER depends on efficient damage recognition. The UvrABC nuclease system, which initiates the NER in Escherichia coli, represents a paradigm for understanding the general mechanism of DNA damage recognition and incision (1). This model system has been widely

Human xeroderma pigmentosum group A (XPA) is an essential protein for nucleotide excision repair (NER). We have previously reported that XPA forms a homodimer in the absence of DNA. However, what oligomeric forms of XPA are involved in DNA damage recognition and how the interaction occurs in terms of biochemical understanding remain unclear. Using the homogeneous XPA protein purified from baculovirus-infected sf21 insect cells and the methods of gel mobility shift assays, gel filtration chromatography, and UV-cross-linking, we demonstrated that both monomeric and dimeric XPA bound to the DNA adduct of N-acetyl-2-aminofluorene (AAF), while showing little affinity for nondamaged DNA. The binding occurred in a sequential and protein concentration-dependent manner. At relatively low-protein concentrations, XPA formed a complex with DNA adduct as a monomer, while at the higher concentrations, an XPA dimer was involved in the specific binding. Results from fluorescence spectroscopic and competitive binding analyses indicated that the specific binding of XPA to the adduct was significantly facilitated and stabilized by the presence of the second XPA in a positive cooperative manner. This cooperative binding exhibited a Hill coefficient of 1.9 and the step binding constants of K 1 = 1.4 × 10 6 M -1 and K 2 ) = 1.8 × 10 7 M -1 . When interaction of XPA and RPA with DNA was studied, even though binding of RPA-XPA complex to adducted DNA was observed, the presence of RPA had little effect on the overall binding efficiency. Our results suggest that the dominant form for XPA to efficiently bind to DNA damage is the XPA dimer. We hypothesized that the concentration-dependent formation of different types of XPA-damaged DNA complex may play a role in cellular regulation of XPA activity.The human XPA (xeroderma pigmentosum group A) protein is an indispensable protein of human nucleotide excision repair (NER) that recognizes and removes a large variety of structurally distinct bulky DNA lesions (1-3). The XPA gene is required for both global genome repair (GGR) and transcription-coupled repair (TCR) (4,5). The protein with a zinc finger motif is capable of binding specifically to the damaged DNA in vitro. XPA interacts with the replication protein A (RPA) to form a protein-protein complex, and this complex formation is believed to be important for NER.A better understanding of the mechanism of human NER, and specifically determination of the role of human XPA, is being actively pursued by research groups including ours. Earlier reports suggest that XPA is involved in the DNA damage recognition process of NER and also † This study was supported by NCI Grant CA86927 (to Y.Z.) and NIEHS Grants ES09127 and ES 00318 (to A.K.B.)