RECQ1 and Ku70/80 co-bind a linear blunt duplex DNA.

<p><i>A.</i> RECQ1 binds a 322 bp blunt duplex fragment derived from pUC19 plasmid DNA. An electrophoretic mobility shift assay (EMSA) was performed to examine the ability of increasing concentration of purified RECQ1 to bind linearized plasmid DNA (30 ng) under DNA binding conditions as described in materials and methods. Protein-DNA complexes were resolved by native 6% polyacrylamide gels and detected by staining with SYBR Gold and a typical inverted image is shown. <i>B.</i> RECQ1 facilitates the formation of higher order DNA complex with Ku70/80. EMSA was performed to examine the ability of increasing concentration of Ku70/80 (0–100 nM) to interact with RECQ1 (12.5 nM) when bound to linear plasmid DNA (30 ng). Both RECQ1 and Ku70/80 bind to blunt duplex resulting in a series of progressively retarded bands, but the mobility of RECQ1-DNA and Ku70/80-DNA complex is distinct. Additional slow migrating bands were observed in Ku70/80 (25, 50 and 100 nM) in the presence of RECQ1 (12.5 nM) (lanes 4–6 and 7–9). <i>C.</i> Molar excess of RECQ1 may compete with Ku70/80 for DNA binding. EMSA was performed to examine the ability of increasing concentration of RECQ1 (0–100 nM) to interact with Ku70/80 (12.5 nM) when bound to linearized plasmid DNA (30 ng) (lane 7 and 8). Arrow indicates the change in band shift pattern of DNA-protein complexes at 50 nM RECQ1. <i>D.</i> RECQ1 and Ku70/80 are co-bound to linear blunt duplex. EMSA reactions were performed using biotinylated DNA probe and the indicated concentration of RECQ1, Ku70/80 or both. The DNA probe was either exposed to a mixture of RECQ1 and Ku70/80, or pre-incubated with one protein followed by addition of the second protein as indicated by parentheses. The DNA-protein complexes were pulled-down on streptavidin magnetic beads and DNA-bound RECQ1 and Ku70/80 were analyzed by Western blotting. Comparable amount of DNA was pull-down in all reactions as shown by agarose gel analyses (bottom). Lane 1 represents biotinylated DNA bound to the streptavidin beads in the absence of RECQ1 or Ku70/80. DNA size marker is shown in leftmost lane of each gel.