Figure 4
Binding of Rif1 to various single-stranded DNAs and their derivatives: stimulation of Rif1 binding by runs of guanine at the 3′-end of the DNA. The single-stranded oligonucleotide DNAs (0.5 pmole) as shown, which had been heat-denatured and renatured in 50 mM KCl and 40% PEG200, were incubated in the presence of increasing amounts of Rif1 protein. With T6G24 and T6(GA)12, 10 fmole (1 nM) of Rif1 was added (+). Samples were analyzed on 12% PAGE (1× 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 topology, as determined by CD analyses (Supplementary Fig. S8), is indicated on top the lanes for each oligonucleotide. The graph shows quantification of the Rif1 binding to each oligonucleotide, conducted as described in the legends to Fig. 2. 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. The quantification of the binding represents the average of three independent experiments with error bars. The background in the absence of Rif1 protein is subtracted. The results of the same, but independent assays are shown in Supplementary Fig. S4.
