Oligonucleotides with Bistranded Abasic Sites Interfere with Substrate Binding and Catalysis by Human Apurinic/Apyrimidinic Endonuclease

Abstract

Apurinic/apyrimidinic endonuclease (AP endo) is a key enzyme in oxidative damage DNA repair. The enzyme, which repairs abasic sites, makes a single nick 5' to the phosphodeoxyribose, leaving a free 3'-hydroxyl. We recently described single turnover kinetics for human recombinant AP endo acting on an oligonucleotide with a single abasic site. We hypothesized that the structural changes induced by the presence of a second abasic site might provide insight into how AP endo recognizes the first abasic site. Here we performed steady state and single turnover experiments using bistranded abasic site substrates, with the second site located on the complementary strand to the one being followed and either opposite to the first or displaced in the 5' direction. All sites on the complementary strand were within half a helical turn of the first. The catalytic efficiency was reduced 80 to 96% and the Kd for substrate binding and dissociation was elevated 40- to 125-fold. The smaller changes occurred when the second site was opposite the first site or displaced by four nucleotides. In addition, if the second abasic site was directly across the helix or displaced by 1 or 3 nucleotides from the first abasic site, cleavage of the first abasic site was subject to apparent substrate inhibition, which did not occur if the second abasic site was displaced by four nucleotides from the first. While a substrate containing a nick without a phosphodeoxyribose on the contralateral strand abasic site did not inhibit nicking of the first strand, a substrate with a nicked abasic site on the contralateral strand was an even stronger inhibitor of enzyme action than an oligonucleotide containing the corresponding abasic site on each strand. Consequently, the inhibitory effect of the second abasic site is probably the result of prior cleavage of the abasic site on the contralateral strand with resulting distortions to the DNA helix that interfere with enzyme binding and/or cleavage.

Substrate Specificity of Human Endonuclease III (hNTH1)

“…The stimulation of hNTH1 activity was independent of the enzyme activity of APE1. The assay mixture lacked MgCl 2 and contained sufficient EDTA to abrogate APE1-mediated endonucleolytic cleavage, but not the binding of APE1 to DNA (18). Identical experiments with Tg:G substrates showed no increase in hNTH1 substrate processing in the presence of APE1 (data not shown).…”

“…Enzyme, protein, and substrate were diluted to working conditions in assay buffer and equilibrated at 37°C. MgCl 2 -independent assays were performed with the above reaction buffer minus MgCl 2 in the presence of 5 mM EDTA, which permits binding of APE1 to DNA, but inhibits its endonuclease activity (18). Reactions contained 40 nM 32 P-5Ј-end-labeled 2Ј-deoxyribose oligonucleotide duplex substrate and 5 nM hNTH1 with or without 20 nM APE1.…”

Repair efficiency of clustered abasic sites by APE1 in nucleosome core particles is sequence and position dependent

“…1 In addition to the endogenously induced $10 000 abasic sites per cell per day, abasic sites are also created exogenously and as intermediates during repair of few other DNA damage lesions. 7,8 Clustered abasic sites are frequently induced by ionizing radiation, chemicals and various other factors. 4-6 A complicated situation is encountered when two or more abasic sites are present in close proximity giving rise to abasic clusters.…”

Induction and Processing of Oxidative Clustered DNA Lesions in56Fe-Ion-Irradiated Human Monocytes

“…Experimental studies suggest much slower rejoining kinetics for DSBs induced by high-LET radiations compared with low-LET radiations (25,26) reflecting the increased complexity of DSBs induced by high-LET radiation (27). Studies with synthetic oligonucleotides containing specific types of clusters suggest that clustered DNA lesions are resistant to processing by glycosylases or endonucleases (28)(29)(30)(31)(32)(33). In vivo studies have shown that the number of abasic clusters formed in human monocytes after irradiation did not return to background levels for over 14 days (14).…”