Figure 3
Binding of Rif1 to various single-stranded DNAs and their derivatives: effect of spacer sequence and runs of guanine on binding of Rif1. The single-stranded oligonucleotide DNAs (0.2 pmole) as shown, which had been heat-denatured and renatured in 50 mM KCl and 40% PEG200, were incubated in the absence (−) or presence (+) of Rif1 protein (10 fmole [1 nM] of the Rif1 full-length polypeptide; the preparation used contains ~10-fold molar excess of degradation products), and were analyzed on 12% PAGE (1x TBE, 50 mM KCl and 40% PEG200). List of oligonucleotides used in the assays and their sequences are shown below the panels. The relevant residues for modification are highlighted in red. The oligonucleotides from which derivatives were made are shown in red. The graph shows quantification of the Rif1 binding to each oligonucleotide with error bars, conducted as described in the legend to Fig. 2. All the binding assays were conducted in separate experiments two times or more with similar results, and one of the representative data are presented. The results of the same, but independent assays are shown in Supplementary Fig. S3. The oligonucleotides showing >50%, >16%, and >6% mobility-shift at 1 nM Rif1 were classified as +++, ++. + for Rif1 binding. M: molecular weight marker (ϕX174 DNA digested by HaeIII). The ticks represent the sizes of 310, 271/281, 234, 194, 118 and 72 bp, from the top.
