Fig. 3
Characterization of GST-CiAPEX2 and GST-CiP0 substrate. a The oligonucleotide sequences used as substrates in each experiment are displayed. 5′ end marked with an asterisk (*) indicates the [γ-32P] ATP-labeled DNA end. In each substrate DNA sequence, underlined bases are the matched or mismatched bases focused on. b and c CiAPEX2 (b) and CiP0 (c) degraded matched DNA more efficiently than mismatched DNA. The reactions were carried out at 28 °C for 60 min using matched or mismatched DNA substrate. Lanes 1 and 5; no protein, Lanes 2-4 and 6-8; investigated proteins. Concentration of investigated proteins were 1 nM (lanes 2 and 6), 10 nM (lanes 3 and 7) and 100 nM (lanes 4 and 8). d Quantification results of the data shown in (b). e Quantification results of the data shown in (c). (f) CiAPEX2 and CiP0 can recognize and degrade nicked DNA, which is generated through THF cleavage by HsAPEX1. As a substrate, THF-containing DNA (Fig. 2a) was fully digested by HsAPEX1 for 60 min. Then, GST-CiAPEX2 or GST-CiP0 was added to the reaction and incubated at 28 °C for 60 min. Lane 1; no protein incubated for 60 min, Lane 2; no protein incubated for 120 min, Lane 3; only HsAPEX1 for 60 min, Lane 4; only HsAPEX1 for 120 min, Lanes 5, 6; after substrate digestion by HsAPEX1 for 60 min, 7.52 pmol GST-CiAPEX2 or 6.84 pmol GST-CiP0 was added and the mixture was incubated for 60 min