Abstract
Base excision repair of oxidized pyrimidines in human DNA is initiated by the DNA N-glycosylase/apurinic/apyrimidinic (AP) lyase, human NTH1 (hNTH1), the homolog of Escherichia coli endonuclease III (Nth). In contrast to Nth, the DNA N-glycosylase activity of hNTH1 is 7-fold greater than its AP lyase activity when the DNA substrate contains a thymine glycol (Tg) opposite adenine (Tg:A) (Marenstein, D. R., Ocampo, M. T. A., Chan, M. K., Altamirano, A., Basu, A. K., Boorstein, R. J., Cunningham, R. P., and Teebor, G. W. (2001) J. Biol. Chem. 276, 21242-21249). When Tg is opposite guanine (Tg:G), the two activities are of the same specific activity as the AP lyase activity of hNTH1 against Tg:A (Ocampo, M. T. A., Chaung, W., Marenstein, D. R., Chan, M. K., Altamirano, A., Basu, A. K., Boorstein, R. J., Cunningham, R. P., and Teebor, G. W. (2002) Mol. Cell. Biol. 22, 6111-6121). We demonstrate here that hNTH1 was inhibited by the product of its DNA N-glycosylase activity directed against Tg:G, the AP:G site. In contrast, hNTH1 was not as inhibited by the AP:A site arising from release of Tg from Tg:A. Addition of human APE1 (AP endonuclease-1) increased dissociation of hNTH1 from the DNA N-glycosylase-generated AP:A site, resulting in abrogation of AP lyase activity and an increase in turnover of the DNA N-glycosylase activity of hNTH1. Addition of APE1 did not abrogate hNTH1 AP lyase activity against Tg:G. The stimulatory protein YB-1 (Marenstein et al.), added to APE1, resulted in an additive increase in both activities of hNTH1 regardless of base pairing. Tg:A is formed by oxidative attack on thymine opposite adenine. Tg:G is formed by oxidative attack on 5-methylcytosine opposite guanine (Zuo, S., Boorstein, R. J., and Teebor, G. W. (1995) Nucleic Acids Res. 23, 3239-3243). It is possible that the in vitro substrate selectivity of mammalian NTH1 and the concomitant selective stimulation of activity by APE1 are indicative of selective repair of oxidative damage in different regions of the genome.
Highlights
Base excision repair of oxidized pyrimidines in human DNA is initiated by the DNA N-glycosylase/ apurinic/apyrimidinic (AP) lyase, human NTH1, the homolog of Escherichia coli endonuclease III (Nth)
Differential Processing of thymine glycol (Tg) Opposite Adenine and Tg Opposite Guanine by human NTH1 (hNTH1)—We previously demonstrated, using a Tg:A-containing substrate, that the initial rate of DNA
N-glycosylase-mediated release of Tg by hNTH1 is much greater than the rate of AP lyase-mediated DNA strand cleavage [1]
Summary
Proteins—Expression and purification of recombinant hNTH1 were induced as described previously [10]. hNTH1 concentration was quantified using an extinction coefficient of 1 absorbance unit at A410 of 69.4 M for the C-terminal cubane 4[Fe-S] cluster [17]. Multiple-turnover assays at Vmax were performed individually in 10-l volumes containing reaction buffer, the indicated concentration of 32P-5Ј-end-labeled 2Ј-deoxyribose oligonucleotide duplex substrate, and 20 nM hNTH1 with or without 100 nM YB-1 and/or 100 nM APE1. To measure strand cleavage and to determine incision products, 5-l aliquots were removed at the indicated time periods, snap-frozen, and treated with an equal volume of 0.5 M NaBH4 at 37 °C for 20 min for reduction of the 2Ј-deoxyribose 5Ј-phosphate moiety. Samples were filtered through a Sephadex G-25 spin column, treated with an equal volume of loading dye, and heated at 55 °C for 5 min, and products were separated by 20% PAGE in 7 M urea and 1ϫ Tris borate/EDTA. All samples were separated by 12% SDS-PAGE and analyzed quantitatively via phosphorimaging as described above
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