In vitro replication and repair studies of tandem lesions containing neighboring thymidine glycol and 8-oxo-7,8-dihydro-2'-deoxyguanosine

Abstract

Reactive oxygen species can induce the formation of tandem DNA lesions. We recently showed that the treatment of calf thymus DNA with Cu2+/H2O2/ascorbate could result in the efficient formation of a tandem lesion where a 5,6-dihydroxy-5,6-dihydrothymidine (or thymidine glycol) is situated on the 5' side of an 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG). In the present study, we assessed how the 5'-Tg-(8-oxodG)-3' and 5'-(8-oxodG)-Tg-3' tandem lesions are replicated by purified DNA polymerases and how they are recognized by base excision repair enzymes. Our results revealed that the tandem lesions blocked primer extension mediated by the Klenow fragment and yeast polymerase eta more readily than when the Tg or 8-oxodG was present alone. The mutagenic properties of Tg or 8-oxodG differed while they were present alone or in tandem. Moreover, the human 8-oxoguanine-DNA glycosylase (hOGG1)-mediated cleavage of 8-oxodG was compromised considerably by the presence of a neighboring 5' Tg, whereas the presence of Tg as the adjacent 3' nucleoside enhanced 8-oxodG cleavage by hOGG1. The efficiency for the cleavage of Tg by endonuclease III was not affected by the presence of an adjoining 8-oxodG. These results supported the notion that the replication and repair of tandem single-nucleobase lesions depend on the types of lesions involved and their spatial arrangement.

Figure 1

The structures of the 5′-Tg-(8-oxodG)-3′ and 5′-(8-oxodG)-Tg-3′ tandem lesions, and the sequences of the 20-mer lesion-containing substrates used in the present in-vitro replication and repair studies.

Figure 2

Primer extension assays for nucleotide incorporation opposite tandem lesions, i.e., 5′-(8-oxodG)-Tg-3′ and 5′-Tg-(8-oxodG)-3′, a single 8-oxodG or Tg, and the undamaged control, with exo^- Klenow fragment (a) and yeast pol η (b). 5′-[³²P]-labeled d(GCTAGGATCATAGC) was used as the primer. Klenow fragment or yeast pol η at the indicated units/concentrations was incubated with 10 nM substrate and 200 μM dNTPs at 37 °C for 60 min. The products were subsequently resolved by using 20% denaturing polyacrylamide gels. The 21-mer was observed due to the presence of a 1-base overhang in the primer, and the 22-mer primer extension products were originated from the terminal transferase activity of the polymerase.

Figure 3

Example gel images for the steady-state kinetic measurements for the nucleotide incorporation opposite the 8-oxodG portion of the 5′-Tg-(8-oxodG)-3′ tandem lesion (a) or the corresponding dG site for the undamaged substrate. (b). Klenow fragment (5 ng) was incubated with 10 nM DNA substrate at room temperature for 10 min. The highest dNTP concentration is shown in the figure, and the ratio of dNTP concentrations between adjacent lanes was 0.5-0.6.

Figure 4

(a) PAGE analysis of the hOGG1-mediated cleavage products of the substrates containing an 8-oxodG, the two tandem lesions, and unmodified GT. “XY” represents different lesions, and the 20 mer 5′-(CA)-3′- and 5′-(AC)-3′-containing complementary strands were used for the repair studies of the 5′-Tg-(8-oxodG)-3′- and 5′-(8-oxodG)-Tg-3′-containing substrates, respectively. (b) A summary of the quantification results of the percent cleavage products for different substrates. The values represent the mean ± standard deviation from three independent treatments and quantification experiments.

Figure 5

(a) PAGE analysis of the products arising from the endonuclease III-mediated cleavage of substrates housing Tg, 5′-Tg-(8-oxodG)-3′, 5′-(8-oxodG)-Tg-3′ and unmodified GT. (b) The quantification results of percent cleavage products for different substrates. The values represent the mean ± standard deviation from three independent treatments and quantification experiments.