Binding of p53 to supercoiled DNA bearing homoadenine-homothymine triplex forming sequences.

<p><b>(A)</b> Scheme of intramolecular T.A.<i>T</i> triplex in scDNA. AFM image of sc pA69 plasmid adsorbed on mica surface in the presence of 2 mM MgCl₂ and complex of pA69 with p53. <b>(B)</b> Comparison of p53 binding to scDNA with and without triplex forming sequence (dA)₅₀.(dT)₅₀ by EMSA. Binding of p53 protein to pBSK, pPGM1, pBA50 and pPA50 detected by EMSA in agarose gel. p53 protein was bound to scDNA (pBSK, 200 ng, lanes 1–5), scDNA with CON (scPGM1, 200 ng, 6–10), scDNA with (dA)₅₀.(dT)₅₀ (scBA50, 200 ng, 11–15) and scDNA with both CON and (dA)₅₀.(dT)₅₀ (scPA50, 200 ng, 16–20) in p53/DNA molar ratios 1–3 at 4°C, EMSA was performed at 4°C. Graph represents the dependence of percents of bound DNA on the amount of p53 proteins calculated from three experiments. <b>(C)</b> Interaction of p53 with scDNA (BSK, PGM1, BA50 and PA50) in presence of pBSK/SmaI (linear competitor, lin) by immunoprecipitation on MBG. Agarose gel electrophoresis of DNA recovered from MBG after incubation of DO1-wtp53-DNA complex at the beads to 50, 100, 300 or 600 mM KCl for 30 min at 10°C followed by the SDS treatment. DNA inputs of scDNA BSK (lane 2), PGM1 (lane 3), BA50 (lane 4), PA50 (lane 5), linBSK (lane 1). Arrows indicate precipitated supercoiled (sc), open circular (oc), linear (lin) and supercoiled dimers (dimer sc). Mean values of bound DNA from three independent experiments were plotted in the graph. Graph represents the dependence of percents of bound DNA on the concentration of KCl in washing buffer calculated from three experiments. <b>(D)</b> Competition assay of p53 binding to CON and non-B-DNA structures in scDNA plasmids. First, full length p53 (60 ng) was incubated with 200 ng PGM1/<i>Pvu</i>II fragments (short fragment with CON sequence (CON, 474 bp) and long fragment as linear nonspecific competitor (NON, 2513 bp) for 20 min on ice to form p53-CON complexes. Subsequently, 200 or 300 ng of different scDNA plasmid competitors were added and incubation was prolonged to 40 min. Plasmids forming triplex T.A.<i>T</i> were marked by TAT, plasmids forming cruciform by X. Graph represents the dependence of percents of bound DNA on the amount of used competitor scDNAs calculated from three experiments.