Figure 3. yCAF1 binding to DNA.

(A) Left panel: Binding curves of yCAF1 variants to 147 bp DNA. Right panels: EMSA showing binding of yCAF1 variants to 147 bp DNA. Free DNA and yCAF1-bound (B) DNA are indicated. Wells (W) are indicated additionally with red horizontal bars. Increasing amounts of yCAF1 (0.15, 0.3, 0.61, 1.25, 2.5, 5 or 10 μM) were mixed with 1 μM DNA. Error bars represent SEM values of three technical replicates (B) Left panel: binding curves of yCAF1 binding to DNA fragments of 20–100 bp (Sequence information in Table 5). Right panel: EMSA showing yCAF1 binding to free DNA fragments of 20–100 bp. Concentration of the DNA 10–100 bp ladder was 275 nM overall nucleotide base pairs present in the binding reaction. yCAF1- DNA binding was quantified by measuring DNA substrate depletion. (C) Left panel: Binding curves of yCAF1 to 17 bp, 42 bp and 84 bp DNA. Right panels: EMSA showing binding of yCAF1 to 17 bp, 42 bp and 84 bp DNA. Wells (W), free DNA and yCAF1-bound (B) DNA are indicated. Increasing amounts of yCAF1 (0.15, 0.3, 0.61, 1.25, 2.5, 5, 10, 20, 40 or 80 μM) were mixed with 1 μM DNA. Error bars represent SEM values of three technical replicates.

DOI: http://dx.doi.org/10.7554/eLife.23474.012

Figure 3—figure supplement 1. Analysis of nucleosome binding by yCAF1.

(A) Anion exchange chromatography of yCAF1 (first peak) revealing a histone-DNA contaminant in the yCAF1 preparation from Trichoplusia ni insect cells (second peak). The identity of all four Trichoplusia ni histones was confirmed by SDS-PAGE and LC-MS. (B) EMSA showing that yCAF1 does not bind to nucleosomes but to free DNA. Increasing amounts of yCAF1 (0.3 μM to 20 μM in two fold steps) were mixed with 2 μM nucleosome. Free DNA, nucleosomes (Nuc), and DNA-bound yCAF1 (B) are indicated. Lane one contained no yCAF1 (-) and lane nine contained yCAF1 but no nucleosome. (C) Uncropped gel image of Figure 3B - EMSA showing yCAF1 binding to free DNA fragments of 20–100 bp.

DOI: http://dx.doi.org/10.7554/eLife.23474.013