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. Author manuscript; available in PMC: 2016 Jan 25.
Published in final edited form as: Cancer Res. 2008 Jun 1;68(11):4142–4149. doi: 10.1158/0008-5472.CAN-08-0796

Figure 1. DNA substrates for εA and 3meC repair and εA repair activities of AlkB and hABH2.

Figure 1

(A) To create εA- and 3meC-containing substrates, 49 mer oligonucleotides were modified with an εA in position 24, or 3meC in position 26, radioactively labeled at the 5’-end and hybridized to a complementary strand. The modified bases are located in a DpnII restriction site such that this enzyme cleaves the substrate only if the etheno/methyl group is removed. Unrepaired substrate appears as a band of 49 nt, whereas repaired and cleaved substrate will appear as a 22 nt band following denaturing PAGE. (B) Activities of purified AlkB, hABH2 and ANPG on εA in ss DNA and ds DNA. DNA substrates were incubated with purified enzymes as indicated, digested with DpnII, or treated with 0.1M NaOH/30 min 90°C if reacted with ANPG, and separated by 20% denaturing PAGE. Labeled DNA was visualized by phosphorimaging. Untreated DNA substrates were incubated with DpnII as negative control. (C) Activities of purified hABH3 on εA in ss DNA and ds DNA. Same reaction conditions as in (B). (D) Reactions as in B. Prior to incubation, cold DNA substrates were added