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. 2004 Jan 16;32(1):397-405.
doi: 10.1093/nar/gkh213. Print 2004.

Nucleotide incorporation by human DNA polymerase gamma opposite benzo[a]pyrene and benzo[c]phenanthrene diol epoxide adducts of deoxyguanosine and deoxyadenosine

Maria A Graziewicz  1 , Jane M Sayer, Donald M Jerina, William C Copeland
Affiliations

Nucleotide incorporation by human DNA polymerase gamma opposite benzo[a]pyrene and benzo[c]phenanthrene diol epoxide adducts of deoxyguanosine and deoxyadenosine

Maria A Graziewicz et al. Nucleic Acids Res. .

Abstract

Mitochondria are major cellular targets of benzo[a]pyrene (BaP), a known carcinogen that also inhibits mitochondrial proliferation. Here, we report for the first time the effect of site-specific N2-deoxyguanosine (dG) and N6-deoxyadenosine (dA) adducts derived from BaP 7,8-diol 9,10-epoxide (BaP DE) and dA adducts from benzo[c]phenanthrene 3,4-diol 1,2-epoxide (BcPh DE) on DNA replication by exonuclease-deficient human mitochondrial DNA polymerase (pol gamma) with and without the p55 processivity subunit. The catalytic subunit alone primarily misincorporated dAMP and dGMP opposite the BaP DE-dG adducts, and incorporated the correct dTMP as well as the incorrect dAMP opposite the DE-dA adducts derived from both BaP and BcPh. In the presence of p55 the polymerase incorporated all four nucleotides and catalyzed limited translesion synthesis past BaP DE-dG adducts but not past BaP or BcPh DE-dA adducts. Thus, all these adducts cause erroneous purine incorporation and significant blockage of further primer elongation. Purine misincorporation by pol gamma opposite the BaP DE-dG adducts resembles that observed with the Y family pol eta. Blockage of translesion synthesis by these DE adducts is consistent with known BaP inhibition of mitochondrial (mt)DNA synthesis and suggests that continued exposure to BaP reduces mtDNA copy number, increasing the opportunity for repopulation with pre-existing mutant mtDNA and a resultant risk of mitochondrial genetic diseases.

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Figures

Figure 1
Figure 1
(A) Structures of the benzo[a]pyrene (BaP) and benzo[c]phenanthrene (BcPh) diol epoxide (DE) enantiomers (diastereomer with a benzylic hydroxyl group and epoxide oxygen trans), as well as their DNA adducts used in this study (dG = dG residue and dA = dA residue with the point of attachment to the hydrocarbon at N2 and N6, respectively). Note that the configuration at the benzylic carbon of the epoxide is inverted on trans opening of the epoxide and retained on cis opening. (B) Template–primer substrates for pol γ with dG and dA adducts at the indicated positions (G* and A*, respectively). A 12mer complementary to the 5′-end of each 22mer primer was annealed to the primer in order to create a substrate with a 22mer primer and a 26mer template containing a gap that does not affect its enzymatic processing. Controls consisted of substrates with the normal (unadducted) bases at positions G* and A*.
Figure 2
Figure 2
Single base insertion opposite normal dG and BaP trans R and trans S dG adducts in oligonucleotides. Reactions containing either Exo–p140 (A) or Exo–p140/p55 complex (B) were incubated for 5 min at 37°C. A partial sequence of the G* templates (Fig. 1B) is shown vertically on both panels of the figure, with the BaP-modified guanine indicated as G**. Lane 0, no dNTPs added; lane 4, all four dNTPs (each at 1 mM final concentration) added; lane T*, 1 µM dTTP (added to initiate a running start one base before the BaP-adducted guanine, G**); lanes A, T, C and G, 1 µM dTTP plus 1 mM one of the four dNTPs. The arrow (B) indicates the products of extension past the adduct.
Figure 3
Figure 3
Single base insertion opposite normal dA and BaP trans R, cis R, trans S and cis S dA adducts in oligonucleotides. Reactions containing either Exo–p140 (A) or Exo–p140/p55 complex (B) were incubated for 5 min at 37°C. A partial sequence of the A* templates (Fig. 1B) is shown vertically on both panels of the figure, with the BaP-modified adenine indicated as A**. Concentrations of the dNTPs and lane designations are as in Figure 3, except that each incubation contained 1 µM dATP to initiate a running start on the A* templates.
Figure 4
Figure 4
Single base insertion opposite normal dA and BcPh cis R and cis S dA adducts in oligonucleotides. Reactions containing either Exo–p140 (A) or Exo–p140/p55 complex (B) were incubated for 5 min at 37°C. A partial sequence of the A* template is shown vertically on both panels of the figure, with the BcPh-modified adenine indicated as A**. Concentrations of the dNTPs and lane designations are as in Figure 3.
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