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. 2019 Mar 14;47(11):e61. doi: 10.1093/nar/gkz171

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© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 7. — Complete repair of an apurinic/apyrimidinic (AP) site or a tetrahydrofuran AP site analog by HepG2 nuclear extract. The data presented in (A) demonstrate that color development observed in the assay plate wells is directly proportional to the amount of fluorescein modified oligonucleotide retained in the wells (r² = 0.99). These data were generated by (i) coating the wells of a Nunc^® Immobilizer™ plate with 0.5 pmol/well of mixtures containing varying proportions of oligonucleotides URA03 (containing a single uracil lesion) and Contr01 (lesion free control), (ii) hybridizing and ligating a complementary oligonucleotide (Loop01Aflc) to the immobilized URA03/Contr01 mixtures to form intact hairpin loop structures covalently attached to the plate at the 3′ end with a fluorescein moiety at the 5′ end, (iii) incubating the immobilized substrates with excess Escherichia coli uracil DNA glycosylase to excise the uracil from those oligonucleotide carrying them to create AP sites, (iv) subjecting the substrate to hot alkaline denaturation to convert all AP sites into single strand breaks and remove any oligonucleotides released as a result and (v) detecting fluorescein retained in the wells using anti-fluorescein horseradish peroxidase conjugate and TMB substrate. Based on this observation, percentage repair was determined by linear interpolation from the absorbance values for 0 and 100% repair control wells. (B) Effects of varying concentrations of HepG2 nuclear extract on the extent of complete AP site repair in the absence (open circles) and presence (closed circles) of excess T4 DNA ligase. (C) Comparison of extent of complete repair by HepG2 nuclear extract of an oligonucleotide complex containing an AP site that had either been pre-treated or not with excess recombinant human AP endonuclease 1. (D) Comparison of the extent of complete repair of an oligonucleotide containing either a normal AP site (black bars) or the tetrahydrofuran AP site analog (white bars) by 800 or 400 ng/well of HepG2 nuclear extract in the presence of either 30 μM of all four deoxynucleotides (dATP, dCTP, dGTP and dTTP) or only 30 μM dTTP. Error bars indicate mean ± SD of triplicate technical replicates.