N2-dG adduct

Benzo[n]pyrene is an extensively studied PAH compound. The mutagenic metabolites of benzo[a]pyrene are the (+)-7R,85,95,101 -flm/-benzo[fl]pyrene-7,8-dihydrodiol-9,10-epoxide, (+)-a //-BPDE, and the (-)-7R,8S,9S,10R enantiomer, ( )-anti-BPDE (Figure 22.16A,B). DNA damage occurs mainly by adduct formation between the CIO position of anti-BPDE to the N2 position of guanine. Four stereoisomeric bulky adducts are produced 105 (+)-trans-anti-BPDE-N2-dG, 1 OR (+)-cis-anti-BPE>E-N2-dG, 1 OR (-)-trans-anti-BPDE-N2-dG, and 105 (-)-cis-anti-BPDE-A2-dG. In vitro, the reaction of (+)-anti-BPDE with DNA forms predominantly the (+)-trans-anti-BPE)E-N2-dG adduct, while the reaction of ( )-anti-BPDE produces mainly the ( )-trans-anti-BPDE-N2-dG adduct. In cells, the major benzo[a]pyrene DNA adduct is (+)-trans-anti-BPDE-N2-dG. 

Purified yeast Pol is able to perform limited nucleotide insertions opposite several DNA lesions such as TT (6-4) photoproduct, AAF-dG adduct, and (+) or ( )-trans-anti-BPDE-N2-dG adduct. Furthermore, Pol also catalyzes extension synthesis from opposite many types of lesions with varying efficiencies, including an AP site, cis-syn TT dimer, (64) photoproduct, AAF-dG adduct, (+) or (-)-trans-an//-BPDE-/V2-dG adduct, and an acrolein-derived dG adduct. Therefore, it has been proposed that Pol functions both as an insertion polymerase and an extension polymerase. It appears that the extension activity of Pol is versatile. Thus, it is believed that Pol is a major extension polymerase during translesion synthesis in eukaryotes. 

All of these B[o]P-N2-dG adducts destabilize the DNA duplexes in a manner that depends on the adduct stereochemistry [53], Detailed thermodynamic studies of some of these adducts [103] and the effects of mismatched bases in the complementary strand opposite all four stereoisomeric B[a]P-N2-dG lesions (G ) in CG C and TG C sequence contexts have been published [104], The melting points of unmodified and B[o]P-modified duplexes are defined as Tm-the temperature at which 50% of the duplexes are dissociated into single strands and are in equilibrium with one another [105]. For convenience, we define the melting points of the modified duplexes by Tm (adduct) and the Tm of the unmodified duplexes by Tm (um). The difference in melting points ATm = Tm(adduct) - Tm (um) is -8 ((+)-trans-), -10 ((-)-trans-), -4 ((+)-ds-), and-5°C ((-)-as-anti-B[a]P-N2-dG) [53],... 

The NER efficiencies in extracts from human HeLa cells of the (+)-trans-, (-)-trans-, and (+)-cis-anti-B[a]P-N2-dG adducts in the identical sequence context (5 -d(CCAT CG CTACC)) (5 -d(GGTAGCGATGG)) embedded in 135mer duplexes are shown in Figure 12.5(a). The lengths of the incision products coincide with those of the marker oligonucleotides around 26-32 nucleotides in length (lane M in Figure 12.5a), which is consistent with dual excision products obtained by the mamma-... 

It is interesting to compare the NER efficiencies of the B[o]P-N2-dG adducts to those of the familiar and widely studied UV-induced thymine-thymine () photodimers. Irradiation of native DNA in vitro or in cells with UV-C light produces isomeric CPD dimers, predominantly the cis-syn form, which involves the covalent finking of two adjacent pyrimidines via a four-membered ring structure (Figure 12.6b). While the CPD dimers do not cause much distortion of double-... 

In order to compare the relative NER efficiencies of our PAH diol epoxide-DNA adducts we prepared site-specifically modified oligonucleotides containing either CPD or 6-4 photodimers using the same sequence and methods as described in the literature [110], Typical results obtained in our laboratory are displayed in Figure 12.6(a). There are fewer NER dual-incision bands in the case of the CPD dimer than in the case of the 6-4 photoproduct and the latter are clearly more intense than the former. Densitometry analysis shows that the time course of dual incisions is linear at least up to 40 min and that the DNA strand bearing the 6-4 adduct is around 17 times faster than the strand with the CPD dimer (Figure 12.6c). The repair rate of the (+)-cis-anti-B[a] P-N2-dG adduct and the 6-4 photoproduct coincide with one another (Figure 12.6c), as described in more detail elsewhere [78],... 

While all three B[a]P-N2-dG adducts destabilize double-stranded DNA, the extent of destabilization is significantly smaller in the case of the (+)-ds-anti adduct (ATm = -4°C) than in the two trans-anti-B[a P-N2-dG adducts (ATm = -8 to —10°C). Nevertheless, the NER efficiency of the (+)-cis-anti-B[a]P-N2-dG adduct is 5-7 times greater than that of either trans-anti adduct. The smaller extent of destabilization caused by the (+)-cis-anti-B[a]P-N2-dG adduct is attributed to carcinogen base n-n stacking interactions that counteract the loss of Watson-Crick hydrogen-bonding and base-base stacking interactions in this base-displaced/intercalated conformation [38]. 

Structural Characteristics of the Identical 10S (+)-trans-ont/-B[a]P-N2-dG Adduct in Different Sequence Contexts... 

The structural properties of B[a]P diol epoxide-derived DNA adducts depend not only on their stereochemical properties as described above, but also on the local base sequence contexts. In order to gain some insights into these phenomena, we focused on the major, biologically significant 10S (+)-tram-anti-B[a]P-N2-dG adduct in the sequence contexts shown in Figure 12.9. We first discuss what is special... 

These are all identical duplexes, except for the following differences the G6G7 and G6 G7 duplexes are identical in composition and 10S (+)-trans-onti-B[a]P-N2-dG adduct stereochemistry except that the lesions are positioned at either of the... 

Dependence ofNER of the 70S (+)-trans-anti-B[aJP-N2-dG Adduct on Base Sequence Context 283... 

The sequence-dependent trend is conserved in the prokaryotic UvrABC system, in which the NER dual-incision efficiency is around 2.4-fold higher for the same 10S (+)-trans-anti-B[a]P-N2-dG adduct in the TG T case than the CG C-II case [124]. Similarly, the G6 G7 duplex is incised more efficiently by a factor of around... 

Figure 12.12 Relative NER efficiencies for the 10S +)-trans-anti-B[a]P-N2-dG adduct embedded in various sequence contexts in otherwise identical 135mer duplexes hybridized with their fully complementary strands.

In this connection, crystal structures of the (+) - trans-anti-1 i a P D E- N2-dG adduct in the Y-family lesion bypass DNA polymerase IV (Dpo4) (see also Chapter 15 by Chandani and Loechler) from the archaeon Sulfolobus solfataricus reveal that the favored P domain is preserved in the enzyme in this case [51], Overall, a survey of crystal structures of DNA damaged by polycyclic carcinogenic chemicals shows that structures observed in DNA duplexes in solution by high-resolution NMR methods are often observed in polymerases [37] however, the preferred P domain can be overridden by strong lesion-polymerase interactions, as was manifested in a crystal structure of a B[a]PDE-derived adenine lesion [52],... 

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