In vitro repair of oxidative DNA damage by human nucleotide excision repair system: possible explanation for neurodegeneration in xeroderma pigmentosum patients

Abstract

Xeroderma pigmentosum (XP) patients fail to remove pyrimidine dimers caused by sunlight and, as a consequence, develop multiple cancers in areas exposed to light. The second most common sign, present in 20-30% of XP patients, is a set of neurological abnormalities caused by neuronal death in the central and peripheral nervous systems. Neural tissue is shielded from sunlight-induced DNA damage, so the cause of neurodegeneration in XP patients remains unexplained. In this study, we show that two major oxidative DNA lesions, 8-oxoguanine and thymine glycol, are excised from DNA in vitro by the same enzyme system responsible for removing pyrimidine dimers and other bulky DNA adducts. Our results suggest that XP neurological disease may be caused by defective repair of lesions that are produced in nerve cells by reactive oxygen species generated as by-products of an active oxidative metabolism.

Figure 1

Substrates used in this study. (a) Structures of the base lesions; dR, deoxyribose. (b) Schematic of duplexes used in the excision assay. Linear duplex molecules, 139 or 140 bp in length, were prepared by phosphorylation, annealing, and ligation of six overlapping oligonucleotides; ∗, position of the radiolabel. (Upper) X denotes 8-OxoG, Tg, or urea.

Figure 2

Excision of oxidative DNA lesions by human and rodent CFEs. (a) Autoradiograph of sequencing gel showing time course of excision by HeLa CFE. In lanes 1 and 4, substrate DNA was resolved without incubation in reaction buffer. (b) Excision of 8-OxoG by CFE from CHO AA8 and its excision repair mutant derivatives. The mutant cell lines used were UV20, UV5, UV24, UV41, and UV135 representative of CG 1–5, respectively. (c) Excision of Tg by CHO AA8 CFE and the same set of mutant cell lines. In a, the entire gel is shown; in b and c, only the region encompassing the excision products is shown. For the 60-min time points with HeLa CFE, the excision products were 0.1% of input DNA (a, lanes 3 and 6) whereas the level of excision with AA8 CFE was ≈2% for 8-OxoG (b, lane 1) and 0.7% for Tg (panel c, lane 1). As with the T<>T substrate (22), the mixture of two mutant CFEs typically gave ≈50% of the wild-type signal. The faint bands visible in lane 6 (c) are due to degradation of the substrate by nucleases in the UV135 CFE.

Figure 3

Time course of excision of oxidative base damage by mammalian excision nuclease using CHO CFEs. Sequencing gels show the results of kinetic experiments conducted as described in the text. (a) The entire gel is shown; (b–d) only the region encompassing the excision products is shown. In the time 0 lanes, substrate DNA was resolved without incubation in reaction buffer. (e) Quantitative analyses of the data shown in a–d and of additional experiments conducted under identical conditions; n = 2–5 experiments except the 90- and 120-min time points with the urea substrate. The bars indicate SEs; in these experiments, 200 amol of excision products represents ≈1.4% excision of input DNA.

Figure 4

Excision of oxidative DNA lesions by reconstituted human excision nuclease. (a) Autoradiograph of sequencing gel showing excision by the XP-dependent reconstituted system. (b) Repair factor omission experiment (25) with 8-OxoG. For these experiments, substrate DNA was resolved in the lanes designated “− Repair Factors” or “All Proteins Omitted” without prior incubation in reaction buffer. (c) For quantitative analysis of excision of oxidative damage with the reconstituted system, the averages of two experiments are plotted. For these experiments, 60 amol of excision products corresponds to ≈0.2% excision of input DNA.