Swi1 facilitates the recruitment of Swi3 to chromatin.

<p>(A, B) ChIP assays of the indicated Swi1 truncated mutants were performed. Swi1 full-length (1–971), Swi1 (250–550), and Swi1 (250–550) precipitate the <i>ori2004</i> regions (<i>ori2004</i> and two positions 14- and 30-kb away from <i>ori2004</i>) in wild-type (WT, in A, middle panels) and <i>swi3</i>Δ mutants (in B, middle panels). Western blotting with anti-FLAG antibodies shows that all FLAG-fused Swi1 truncations mutants were similarly expressed (top panels). Fold increase in chromatin association over background (FLAG, set to 1) was calculated for each band. The average fold increase in the association of Swi1 truncations with the three positions (<i>ori200</i>4, −14 kb, and −30 kb) is shown, and error bars represent standard deviations obtained from the three positions (bottom panels). Representative results of repeat experiments are shown. WCE, whole cell extract; WB, Western blotting. (C) ChIP assay of GST-Swi3 was performed in wild-type or <i>swi1</i>Δ cells expressing the indicated proteins. GST-Swi3 strongly associates with the <i>ori2004</i> region in wild-type (WT), while GST-Swi3 has weak chromatin association in the absence of Swi1. Western blotting with anti-GST antibodies shows that GST-Swi3 was expressed similarly in wild-type and <i>swi1</i>Δ cells. Quantification of PCR bands was performed as described above. Fold increase in chromatin association over background (GST, set to 1) in each cell line (WT or <i>swi1</i>Δ) is shown. Representative results of repeat experiments are shown. (D) In vitro DNA binding assays of Swi1 (235–564) and Swi3 purified from the indicated <i>S. pombe</i> cells (top panel). The indicated proteins were mixed with radiolabeled plasmid pUC28, and associated DNA was analyzed by agarose gel electrophoresis (middle panel). Quantification of bound DNA was performed as described in Materials and Method. The values of bound DNA were normalized to the amount of proteins used in the reactions, and relative levels of bound DNA over background (GST) are shown. Representative results of repeat experiments are shown. C, input radiolabeled DNA.