The C-clamp of POP-1 facilitates binding to DNA containing Helper sites.

Abstract

<p>(A–D) EMSAs showing binding of wild-type recombinant POP-1 and a POP-1 C-clamp mutant to the <i>ceh-22b</i> WRE probe (1.5 femtomoles/reaction) described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004133#pgen-1004133-g001" target="_blank">Figure 1A</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004133#pgen-1004133-g002" target="_blank">2N</a> (A), the <i>psa-</i>3 probe (3 femtomoles/reaction) described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004133#pgen-1004133-g001" target="_blank">Figure 1B</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004133#pgen.1004133.s001" target="_blank">S1B</a> (B), the <i>K08D12.3</i> probe (4 femtomoles/reaction) described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004133#pgen-1004133-g003" target="_blank">Figure 3A and 3F</a> (C) and the <i>ceh-22b</i> probe (3 femtomoles/reaction) with both Helper sites mutated (D). The <i>ceh-22b</i>, <i>psa-3</i> and <i>K08D12.3</i> WT probes show strong binding with increasing amounts (0.4 and 0.8 µg/reaction) of POP-1 WT protein (lanes 2 and lane 3 respectively) but not with the POP-1 C-clamp mutant (lane 4 and lane 5 respectively). Under conditions designed to detect lower affinity binding (0.75 and 1.5 µg of POP-1; 3 femtomoles of probe and longer exposure times), binding to the <i>ceh-22b</i> probe lacking Helper sites (containing only the HMG2 site) was similar with WT and mutant POP-1. The data are representative of three independent experiments.</p