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. 2015 Jun 29;24(18):5184–5197. doi: 10.1093/hmg/ddv240

Figure 4.

Figure 4.

The WT/G451E p55 heterodimer has weakened binding affinities for dsDNA and for p140. (A) Determination of the dissociation constants for DNA binding, Kd(DNA). Representative binding isotherms for WT p55 (open circle) and WT/G451E (closed circle) p55 binding to end-labeled double-stranded oligonucleotides are presented. Reactions (20 µl) contained 15 nM dsDNA and varying amounts of accessory subunits. DNA-binding reactions were resolved by EMSAs as described in Materials and Methods. (B) Determination of the dissociation constants for p140 binding, Kd(p140). Representative binding isotherms for accessory subunits binding to p140 as measured by DNA polymerase activity on poly(dA)•p(dT)12–18 are presented. Reactions (50 µl) contained 1 nM p140 and varying amounts of WT (open circle) or WT/G451E (closed circle) p55 heterodimer. Experiments were repeated twice independently. The dissociation constants were calculated by quadratic curve fitting as described in Materials and Methods.