Comparative Mutagenesis of the C8-Guanine Adducts of 1-Nitropyrene and 1,6- and 1,8-Dinitropyrene in a CpG Repeat Sequence

Abstract

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 (8)-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 repaircompetent 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. 1-Nitropyrene (1-NP)1 and the dinitropyrenes are common environmental pollutants (1-3). Most of the nitropyrenes are mutagenic (for a review see Ref. 4) and tumorigenic (5-8), but their potency sometimes differs by more than an order of magnitude. Nitroreduction is a major pathway of bioactivation of all nitropyrenes, whereas O-esterification enzymes, in addition, play a crucial role in the mutagenicity of DNPs.1-NP and the DNPs revert Salmonella typhimurium frameshift tester strains TA98 and TA1538 more efficiently than the strains TA100 and TA1535 that detect base pair substitutions (9). The most frequent mutation among the revertants in TA98 is a two-base deletion of a GpC or CpG pair within a CGCGCGCG hotspot sequence upstream of the hisD3052 mutation (10). The frequency of reversion induced by 1-NP in TA98 drops sharply in TA98NR that lacks the classical nitroreductase (11). By contrast, the frequency of reversion by 1,6-and 1,8-DNP is only slightly lower in TA98NR but is significantly reduced in TA98...

Replicative Bypass of Abasic Site in Escherichia coli and Human Cells: Similarities and Differences

“…Construction of the modified and control M13 genome involved digestion of M13mp7L2 single-stranded DNA with Eco RI, annealing a 50-mer scaffold, and ligation of the Z-containing and control 12-mer, which followed the protocol described earlier in detail [31]. The efficiency of ligation was ∼40% for both the control and modified 12-mer.…”

“…Unligated oligonucleotides were removed by passing through Centricon-100 and the DNA was precipitated with ethanol. The scaffold removal followed the same protocol as described [31], [32]. The final construct was dissolved in 1 mM Tris-HCl-0.1 mM EDTA, pH 8, and a portion was subjected to electrophoresis on 1% agarose gel in order to assess the amounts of circular DNA.…”

Mutagenicity of the 1-Nitropyrene-DNA Adduct N-(Deoxyguanosin-8-yl)-1-aminopyrene in Mammalian Cells

“…In E. coli the site-specifically situated C8-AP-dG adduct in the CGCGCG sequence induced two-base deletions (22,23), whereas both base substitutions and one-base deletions occurred in the non-repetitive CCATCGCTACC sequence (24). In each case the adduct is flanked by a 5′ and a 3′ C, yet the mutations in these two sites are very different in E. coli.…”

“…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.…”

Frameshift mutations induced by four isomeric nitroacridines and their des-nitro counterpart in thelacZ reversion assay inEscherichia coli

“…it explains À1 mutagenesis in a monotonous run (e.g., GGGGG), except that there is a dinucleotide bulge rather than a single bulged base [Koffel-Schwartz and Fuchs, 1995;Shelton and DeMarini, 1995;Hoffmann and Fuchs, 1997;Hilario et al, 2002;Hoffmann et al, 2003]. Studies of AAF show that adducts on the C8 position of guanine at sites of alternating base pairs stabilize a slipped intermediate [Garcia et al, 1993;Milhé et al, 1994Milhé et al, , 1996Hoffmann and Fuchs, 1997].…”