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. 2012 Sep 19;287(46):39233–39244. doi: 10.1074/jbc.M112.369975

FIGURE 2.

FIGURE 2.

Characterization of DNA ligase IIIα-XRCC1 complexes containing either wild-type XRCC1 or the A482T mutant. A, purified DNA ligase IIIα-XRCC1 complexes (150 ng) containing wild-type XRCC1 (lane 1) and the A482T mutant (lane 2). Proteins were separated by SDS-PAGE and then stained with Coomassie Blue. The bands corresponding to DNA ligase IIIα (LigIII) and XRCC1 (XRCC1) are indicated. B, the joining of a labeled nicked DNA substrate (upper panel and middle panel, lane 1) by DNA ligase IIIα-XRCC1 complexes containing either wild-type XRCC1 (WT, middle panel, lanes 2 and 3) or the A482T mutant (Mut, middle panel, lanes 4 and 5) was measured as described under “Experimental Procedures.” The results of three independent experiments are shown graphically (lower panel). C, upper panel, the binding of DNA ligase IIIα-XRCC1 complexes containing either wild-type XRCC1 (XRCC1-WT) or the A482T mutant (XRCC1-MutPNK482) to magnetic Ni2+ beads with (lanes 3 and 6) or without His-tagged PNKP (lanes 2 and 5). Pulldown assays were carried out as described under “Experimental Procedures.” 10% input of DNA ligase IIIα-XRCC1 complexes with: lane 1, wild-type XRCC1 and lane 4, A482T mutant version. XRCC1 was detected by immunoblotting. The results of two independent experiments are shown graphically with the error bars representing S.D. (lower panel). The binding of XRCC1 is expressed as a percentage of the binding by wild-type XRCC1.