HMG I(Y) effects on p53 complexes with Holliday junction and 3-cytosine bulge probes. (A) Dissociation of p53/Holliday junction complexes by HMG I(Y). Holliday junction and duplex probes (7.5 nM) were incubated with either p53, HMG I(Y) or both proteins at room temperature for 20 min. The reaction products were separated by non-denaturing polyacrylamide gel electrophoresis followed by autoradiography. The concentrations of proteins used are indicated above each lane. Arrows and brackets indicate the positions of the probe, p53/DNA complexes and HMG I(Y)/DNA complexes. (B) Pre-formed p53/Holliday junction complexes are also dissociated by HMG I(Y). Holliday junction probes (7.5 nM) were pre-incubated with p53 (115 nM) for 20 min followed by the addition of HMG I(Y) (20 nM) for increasing amounts of time. The reaction products were analyzed by non-denaturing polyacrylamide gel electrophoresis. (C) The rate of dissociation of p53/Holliday junction complexes is similar at three concentrations of p53 tested. Holliday junction probes were incubated with 34 nM (circles), 75 nM (squares) or 115 nM p53 (triangles) in the presence of increasing amounts of HMG I(Y) followed by gel retardation assays. p53/DNA complexes were quantified, normalized to complex formation in the absence of HMG I(Y), and plotted as a function of HMG I(Y) concentration.

HMG I(Y) effects on hMSH2–hMSH6 complexes from Holliday junction and 3-cytosine bulge templates. (A) HMG I(Y) dissociates hMSH2–hMSH6 complexes from Holliday junctions and 3-cytosine bulges. Holliday junction, duplex or 3-cytosine bulge probes (7.5 nM) were incubated with hMSH2–hMSH6, HMG I(Y) or both proteins at room temperature for 20 min. The reaction products were separated by non-denaturing polyacrylamide gel electrophoresis followed by autoradiography. Protein concentrations are indicated above each lane. Arrows and brackets indicate the positions of the probe, hMSH2–hMSH6/DNA complexes and HMG I(Y)/DNA complexes. (B) HMG I(Y) causes dissociation of hMSH2–hMSH6 complexes from Holliday junction probes at the same rate as the 3-cytosine bulge probes. Probes containing 3-cytosine bulges or Holliday junctions (7.5 nM) were incubated with 75 nM hMSH2–hMSH6 in the presence of increasing amounts of HMG I(Y) followed by gel retardation assays. Protein/DNA complexes were quantified and plotted as a function of HMG I(Y) concentration.

HMG-1 does not significantly affect p53 binding to Holliday junctions. Holliday junction probes (7.5 nM) were incubated with p53, HMG I(Y) or both proteins at room temperature for 20 min. The reaction products were separated by non-denaturing polyacrylamide gel electrophoresis followed by autoradiography. Lanes 1 and 15, probe; lanes 2–7 and 16: 75 nM p53; lanes 8–13, 115 nM p53, lanes 3 and 9, 10 nM HMG1; lanes 4 and 10, 20 nM HMG1; lanes 5 and 11, 40 nM HMG1; lanes 6 and 12, 80 nM HMG1; lanes 7 and 13, 170 nM HMG1; lanes 14 and 17, 17 nM HMG1. Arrows and brackets indicate the positions of the probe, p53/DNA complexes and HMG1/DNA complexes.

Effect of HMG I(Y) on p53 binding to 3-cytosine bulge containing templates. (A) HMG I(Y) has no significant effect on p53 binding to DNA containing 3-cytosine bulges. The 3-cytosine bulge probe (7.5 nM) was incubated with 75 nM p53 and increasing amounts of HMG I(Y) and products analyzed by gel retardation assays. Protein concentrations are indicated above the lanes. (B) p53 complexes with the 3-cytosine bulge probe remain constant at all concentrations of HMG I(Y) tested. Probes containing 3-cytosine bulges (7.5 nM) were incubated with 75 nM (circles) or 115 nM p53 (squares) in the presence of increasing amounts of HMG I(Y) followed by gel retardation assays. p53/DNA complexes were quantified and plotted as a function of HMG I(Y) concentration.

hMSH2–hMSH6 effect on p53 binding to Holliday junction and 3-cytosine bulge templates. (A) hMSH2–hMSH6 induces p53 complex formation with Holliday junction substrates. Holliday junction probes (7.5 nM) were incubated with either p53 or hMSH2–hMSH6 or both proteins at concentrations indicated above each lane and complexes separated from free probe by gel retardation assays. (B) Quantification of p53 complexes with Holliday junctions in the presence of hMSH2–hMSH6. Holliday junction probes (7.5 nM) were incubated with 75 nM p53 and 0, 17, 34, 51 and 65 nM hMSH2–hMSH6 and complexes separated by gel retardation assays (n = 4). p53/DNA complexes were quantified and the fold increase in complex formation was plotted as a function of hMSH2–hMSH6 concentration. (C) hMSH2–hMSH6 induces p53 complex formation with 3-cytosine bulges. Holliday junction probes (7.5 nM) were incubated with either p53 or hMSH2–hMSH6 or both proteins at concentrations indicated above each lane and complexes separated from free probe by gel retardation assays.