Skip to main content
Fig. 2 | Genes and Environment

Fig. 2

From: CiAPEX2 and CiP0, candidates of AP endonucleases in Ciona intestinalis, have 3′-5′ exonuclease activity and contribute to protection against oxidative stress

Fig. 2

Enzymatic characterization of GST-CiAPEX2 and GST-CiP0. a-c The oligonucleotide sequences used as substrates in each experiment are displayed. In these sequences, F and X represent tetrahydrofuranyl (THF)-AP site analogue and thymine glycol (Tg). The 5′ end marked with an asterisk (*) indicates the [γ-32P] ATP-labeled DNA end. d and e Detection of AP endonuclease activity for GST-CiAPEX2 (d) and GST-CiP0 (e). The reaction was carried out at 28 °C for 20 min using DNA substrate shown in (a). Lane 1, no protein; Lane 2, 1 unit HsAPEX1(New England BioLabs); Lanes 3-5, investigated proteins. Concentration of investigated proteins were 1 nM (lane 3), 10 nM (lane 4) and 100 nM (lane 5). f and (g Detection of 3′-phosphodiesterase activity for GST-CiAPEX2 (f) and GST-CiP0 (g). The reaction was carried out at 28 °C for 20 min using DNA substrate shown in (b). The β-products generated by GST-CiNTH give rise to two separate bands, presumably because of Tris-adduct formation [33, 34] or isomerization of the 3′-hydroxypentenal terminus [35, 36]. Lane 1, no protein; Lane 2, GST-CiNTH alone; Lanes 3-5, GST-CiNTH and GST-CiAPEX1; Lanes 6-8, GST-CiNTH and investigated protein. Concentration of added CiNTH was constant at 1 nM. Concentration of other added proteins were as follows, 1 nM (lanes 3 and 6), 10 nM (lanes 4 and 7) and 100 nM (lanes 5 and 8). h and j Detection of 3′-5′ exonuclease activity for GST-CiAPEX2 (h) and GST-CiP0 (j). The reaction was carried out at 28 °C for 60 min using DNA substrate shown in (c). Lane 1, no protein; Lanes 2-4, investigated proteins. Concentration of investigated proteins were 1 nM (lane 2), 10 nM (lane 3) and 100 nM (lane 4). i and k 3′-5′ exonuclease activity of CiAPEX2 and CiP0 are dependent on metal ion. Chelating agent, EDTA, was added to the reaction buffer as indicated. Lane 1; no protein, lane 2; purified protein only, lane 3; purified protein and 10 mM EDTA, lane 4; purified protein and 20 mM EDTA

Back to article page