For assays involving 6-FAM labeled DNA substrates, purified Ctp1 proteins were incubated with 10 nM DNA substrate in 1X reaction buffer (20 mM Tris pH 7.5, 25 mM NaCl, 0.1 mM DTT, 10 µg ml−1 bovine serum albumin, 0.5% glycerol), for 20 minutes at 20°C. Assays examining mutants of the C-terminus (Fig. 4e ) used 200 nM protein in each reaction. Reactions were resolved on Native PAGE 4–20% gradient TBE gels (Invitrogen) on ice and imaged using a Typhoon 9000 imager (GE Healthcare). For Ctp1 binding a 10 bp DNA ladder (Supplementary Fig.5b ), purified Ctp1 was incubated with 1 µg µl−1 of a 10bp DNA ladder (Invitrogen), in 1X reaction buffer for 20 minutes at room temperature. Reactions were resolved on a 3% TBE agarose gel. Uncropped gels are available in Supplementary Data Set 1 .
For quantitative DNA binding studies (Supplementary Fig. 5a ), DNA binding and gel electrophoresis were carried out at 4°C. DNA binding experiments (n=3 for each DNA substrate) were analyzed using ImageJ (http://imagej.nih.gov/ij/ ). Dissociation constants were calculated using one-site specific binding with hill slope in GraphPad Prism 6.0a (GraphPad Software, Inc.).
For quantitative DNA binding studies (