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. 2002 Dec 1;30(23):5284-92.
doi: 10.1093/nar/gkf643.

Translesion replication of benzo[a]pyrene and benzo[c]phenanthrene diol epoxide adducts of deoxyadenosine and deoxyguanosine by human DNA polymerase iota

Ekaterina G Frank  1 , Jane M Sayer, Heiko Kroth, Eiji Ohashi, Haruo Ohmori, Donald M Jerina, Roger Woodgate
Affiliations

Translesion replication of benzo[a]pyrene and benzo[c]phenanthrene diol epoxide adducts of deoxyadenosine and deoxyguanosine by human DNA polymerase iota

Ekaterina G Frank et al. Nucleic Acids Res. .

Abstract

Human DNA polymerase iota (poliota) is a Y-family polymerase whose cellular function is presently unknown. Here, we report on the ability of poliota to bypass various stereoisomers of benzo[a]pyrene (BaP) diol epoxide (DE) and benzo[c]phenanthrene (BcPh) DE adducts at deoxyadenosine (dA) or deoxyguanosine (dG) bases in four different template sequence contexts in vitro. We find that the BaP DE dG adducts pose a strong block to poliota-dependent replication and result in a high frequency of base misincorporations. In contrast, misincorporations opposite BaP DE and BcPh DE dA adducts generally occurred with a frequency ranging between 2 x 10(-3) and 6 x 10(-4). Although dTMP was inserted efficiently opposite all dA adducts, further extension was relatively poor, with one exception (a cis opened adduct derived from BcPh DE) where up to 58% extension past the lesion was observed. Interestingly, another human Y-family polymerase, polkappa, was able to extend dTMP inserted opposite a BaP DE dA adduct. We suggest that poliota might therefore participate in the error-free bypass of DE-adducted dA in vivo by predominantly incorporating dTMP opposite the damaged base. In many cases, elongation would, however, require the participation of another polymerase more specialized in extension, such as polkappa.

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Figures

Figure 1
Figure 1
Structures of BaP and BcPh and their diol epoxide (DE) metabolites, with derived DNA adducts (Base = dG or dA residue with the point of attachment at N2 of G or N6 of dA as indicated). Only adducts derived from the DE shown (two enantiomers of the diastereomer [DE-2 or ‘anti’ DE] in which the benzylic hydroxyl group and the epoxide oxygen are trans) were used in this study. The angular benzo ring of the hydrocarbon that is metabolized to the DE is shown in bold. Note that for cis adducts, the configuration at the benzylic C-10 or C-1 of the epoxide is retained, whereas for trans adducts it is inverted.
Figure 2
Figure 2
Ability of polι to replicate two stereoisomeric BaP DE dG adducts in two sequence contexts (A and B). For each panel, the sequence of the primer–template used in the reaction is shown above the reactions. Reactions were performed for 30 min at 37°C in the presence of the four dNTPs (4) at 100 µM each or individually, G, A, T and C (at 100 µM). The template either contained no lesion or a G adduct indicated by an asterisk in the template.
Figure 3
Figure 3
Ability of polι to replicate four stereoisomeric BaP DE dA adducts in two sequence contexts (A and B). For each panel, the sequence of the primer–template used in the reaction is shown above the reactions. Reactions were performed for 30 min at 37°C in the presence of the four dNTPs (4) at 100 µM each or individually, G, A, T and C (at 100 µM). The template either contained no lesion or an A adduct indicated by an asterisk in the template.
Figure 4
Figure 4
Ability of polι to replicate two stereoisomeric BcPh DE dA adducts in two sequence contexts (A and B). For each panel, the sequence of the primer–template used in the reaction is shown above the reactions. Reactions were performed for 30 min at 37°C in the presence of the four dNTPs (4) at 100 µM each or individually, G, A, T and C (at 100 µM). The template either contained no lesion or a dA adduct indicated by an asterisk in the template.
Figure 5
Figure 5
Comparison of the ability of human polι and rat polβ to bypass four stereoisomeric BaP DE dA adducts or two stereoisomeric BcPh DE dA adducts in two sequence contexts (A and B). The sequence of the primer– template used in the reactions is shown above their respective panels. Reactions were performed for 30 min at 37°C in the presence of the four dNTPs at 100 µM each; in these assays, KCl was omitted from the reactions and DTT was replaced with 10 mM β-mercaptoethanol. Lane 1, radiolabeled primer (no template); lane 2, no lesion; lane 3, BaP trans R; lane 4, BaP trans S; lane 5, BaP cis R; lane 6, BaP cis S; lane 7, BcPh cis R; lane 8, BcPh cis S.
Figure 6
Figure 6
Ability of human polκ to misincorporate bases opposite a BaP DE trans S dA lesion and extend polι-dependent incorporation of dTMP opposite BaP DE trans S dA. The sequence of the 16mer primer/29mer BaP DE dA template used in the reactions is shown above the replication assays. The location of the adduct is indicated by an asterisk. In these assays, KCl was omitted from the reactions and DTT was replaced with 10 mM β-mercaptoethanol. Reactions were performed for the times noted at 37°C in the presence of the four dNTPs (4) at 100 µM each or individually, G, A, T and C (at 100 µM). The concentration of polι was kept constant at 4 nM, while the concentration of polκΔC was varied as noted above each reaction. In the case of the polι plus polκ reactions, 4 nM polι was allowed to extend the 16mer primer annealed to the damaged template in the presence of all four dNTPs (100 µM each) for 10 min before the subsequent addition of various concentrations of polκΔC for an additional 10 min. As a consequence, the total reaction time indicated on the figure for polι is 20 min.
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References

    1. Thakker D.R., Yagi,H., Levin,W., Wood,A.W., Conney,A.H. and Jerina,D.M. (1985) Polycyclic aromatic hydrocarbons: metabolic activation to ultimate carcinogenesis. In Anders,M.W. (ed.), Bioactivation of Foreign Compounds. Academic Press, New York, NY, pp. 177–242.
    1. Jerina D.M., Sayer,J.M., Agarwal,S.K., Yagi,H., Levin,W., Wood,A.W., Conney,A.H., Pruess-Schartz,D., Baird,W.M., Pigott,M.A. and Dipple,A. (1986) Reactivity and tumorigenicity of bay-region diol epoxides derived from polycyclic aromatic hydrocarbons. In Kocsis,J.J., Jollow,D.J., Witmer,C.M., Nelson,J.O. and Snyder,R. (eds), Biological Reactive Intermediates. Plenum Press, New York, NY, Vol. 3. pp. 11–30. - PubMed
    1. Jerina D.M., Yagi,H., Thakker,D.R., Sayer,J.M., van Bladeren,P.J., Lehr,R.E., Whalen,D.L., Levin,W., Chang,R.L., Wood,A.W. and Conney,A.H. (1984) Identification of ultimate carcinogenic metabolites of the polycyclic aromatic hydrocarbons: bay-region (R,S)-diol-(S,R)-epoxides. In Caldwell,J. and Paulson,G.D. (eds), Foreign Compound Metabolism. Taylor & Francis, London, UK, pp. 257–266.
    1. Jerina D.M., Chadha,A., Cheh,A.M., Schurdak,M.E., Wood,A.W. and Sayer,J.M. (1991) Covalent bonding of bay-region diol epoxides to nucleic acids. In Witmer,C.M., Snyder,R., Jollow,D.J., Kalf,G.F., Kocsis,J.J. and Sipes,I.G. (eds), Biological Reactive Intermediates IV. Molecular and Cellular Effects and Their Impact on Human Health. Plenum Press, New York, NY, pp. 533–553. - PubMed
    1. Cheng S.C., Hilton,B.D., Roman,J.M. and Dipple,A. (1989) DNA adducts from carcinogenic and noncarcinogenic enantiomers of benzo[a]pyrene dihydrodiol epoxide. Chem. Res. Toxicol., 2, 334–340. - PubMed

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