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

FIGURE 1.

FIGURE 1.

XRCC1 domain structure and regions involved in protein-protein interactions; replacement of alanine at position 482 with threonine disrupts the binding of XRCC1 to PNKP but not aprataxin or APLF. A, diagram showing the positions of the nuclear localization signal (NLS), N-terminal domain (NTD), and the two BRCT domains (BRCT I and BRCT II) within XRCC1. The regions involved in binding to APE1 (20), PARP1 (23), Polβ (13), PNKP (26), and DNA ligase IIIα (9, 11) and the positions of the amino acid changes that disrupt binding to APE1 (G297V), PARP1 (L316R), Polβ (A160S), PNKP (A482T), and DNA ligase IIIα (P623S) are indicated. B, the effect of replacing alanine with threonine (pGADT7-MutPNK482) at position 482 on the interaction of XRCC1 with PNKP (pGBK-PNK) and DNA ligase IIIα (pGBK-LigIII) was analyzed using the yeast two-hybrid assay. C, the effect of replacing alanine 482 with threonine on the interaction of XRCC1 with PNKP, aprataxin, and APLF. Upper panel, the binding of purified PNKP (PNKP) to glutathione beads liganded by GST (GST), GST fused to wild-type XRCC1 (GST-WT) or GST fused to the A482T mutant version of XRCC1 (pGADT7-MutPNK482). Middle panel, the binding of labeled in vitro translated wild-type XRCC1 (WT) and the A482T mutant version of XRCC1 (MutPNK482) to glutathione beads liganded by GST (GST) and GST fused to APLF (GST-APLF). Lower panel, the binding of labeled in vitro translated wild-type XRCC1 (WT) and the A482T mutant version of XRCC1 (MutPNK482) to glutathione beads liganded by GST (GST) and GST fused to aprataxin (GST-Aprataxin). Pulldown assays were carried out as described under “Experimental Procedures.”