Klenow Fragment Discriminates against the Incorporation of the Hyperoxidized dGTP Lesion Spiroiminodihydantoin into DNA

Abstract

Defining the biological consequences of oxidative DNA damage remains an important and ongoing area of investigation. At the foundation of understanding the repercussions of such damage is a molecular-level description of the action of DNA-processing enzymes, such as polymerases. In this work, we focus on a secondary, or hyperoxidized, oxidative lesion of dG that is formed by oxidation of the primary oxidative lesion, 2'-deoxy-8-oxo-7,8-dihydroguanosine (8-oxodG). In particular, we examine incorporation into DNA of the diastereomers of the hyperoxidized guanosine triphosphate lesion spiroiminodihydantoin-2'-deoxynucleoside-5'-triphosphate (dSpTP). Using kinetic parameters, we describe the ability of the Klenow fragment of Escherichia coli DNA polymerase I lacking 3' → 5' exonuclease activity (KF(-)) to utilize (S)-dSpTP and (R)-dSpTP as building blocks during replication. We find that both diastereomers act as covert lesions, similar to a Trojan horse: KF(-) incorporates the lesion dNTP opposite dC, which is a nonmutagenic event; however, during the subsequent replication, it is known that dSp is nearly 100% mutagenic. Nevertheless, using kpol/Kd to define the nucleotide incorporation specificity, we find that the extent of oxidation of the dGTP-derived lesion correlates with its ability to be incorporated into DNA. KF(-) has the highest specificity for incorporation of dGTP opposite dC. The selection factors for incorporating 8-oxodGTP, (S)-dSpTP, and (R)-dSpTP are 1700-, 64000-, and 850000-fold lower, respectively. Thus, KF(-) is rigorous in its discrimination against incorporation of the hyperoxidized lesion, and these results suggest that the specificity of cellular polymerases provides an effective mechanism to avoid incorporating dSpTP lesions into DNA from the nucleotide pool.

Figure 1

Structures of dG, 8-oxodG, (S)-dSp, and (R)-dSp. The A and B rings of dSp are indicated.

Figure 2

(A) Sequence of primer and template strands. Primer was ³²P-radiolabeled at 5'-end as indicated by the asterisk. (B) Primer extension on different template DNA (X = A, C, G or T) with either dGTP, 8-oxodGTP, (S)-dSpTP, or (R)-dSpTP. The five lanes on the left in panel B are the primer and the positive controls with correct nucleotide for each template DNA. (C) Primer extension after the incorporation of dSpTP. See Experimental Procedures for reaction conditions.

Figure 3

Kinetic analysis of dGTP and 8-oxodGTP incorporation by KF⁻. Enzyme model used for (A) dGTP and (B) 8-oxodGTP. (C) Global fitting of incorporation of dGTP opposite of template dC. The concentrations of dGTP are 0.5 µM (closed circles), 1 µM (open circles), 10 µM (closed squares), 50 µM (open squares), and 100 µM (closed triangles). (D) Global fitting of incorporation of 8-oxodGTP opposite of template dC. The concentrations of 8-oxodGTP are 1 µM (closed circles), 2 µM (open circles), 5 µM (closed squares), 10 µM (open squares), and 50 µM (closed triangles). The curves superimposed with the experimental data were generated by KinTek Explorer fitting.

Figure 4

Kinetic analysis of (S)-dSpTP and (R)-dSpTP incorporation by KF⁻. (A) Enzyme model used for global fitting. (B) Global fitting of incorporation of (S)-dSpTP opposite of template dC. (S)-dSpTP concentrations are 10 µM (closed circles), 20 µM (open circles), 50 µM (closed squares), 100 µM (open squares), and 200 µM (closed triangles). (C) Global fitting of incorporation of (R)-dSpTP opposite of template dC. (R)-dSpTP concentrations are 50 µM (closed circles), 100 µM (open circles), 200 µM (closed squares), 400 µM (open squares), and 600 µM (closed triangles). The curves superimposed with the experimental data were generated by KinTek Explorer fitting. FitSpace analysis by KinTek Explorer showing the results of the initial excursions to map the boundaries of a good fit for (D) (S)-dSpTP and (E) (R)-dSpTP.

Figure 5

Analysis of dGTP (open triangles), 8-oxodGTP (closed circles), (S)-dSpTP (open squares), and (R)-dSpTP (closed triangles) kinetic results using Michaelis-Menten techniques where the observed rates are plotted against nucleotide concentrations. The insert is a zoomed out view of the main panel and allows for comparison of dGTP to the three oxidized nucleotides.