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. 2013 Jun 6;50(5):661–674. doi: 10.1016/j.molcel.2013.05.011

Figure 2.

Figure 2

DDK and Sld3–Sld7 Localize at Centromeric Regions from Telophase to G1 Phase

(A) DDK and Sld3–Sld7 localize at centromeric regions from telophase to G1 phase. Cells with CDC7-GFP MTW1-CFP NDC80-CFP SPC42-mCherry NIC96-mCherry (T10876 [i]), GFP-DBF4 MTW1-CFP NDC80-CFP (T9466 [ii]), SLD7-GFP NDC80-CFP SPC42-mCherry NIC96-mCherry (T10875 [iii]), and SLD3-GFP SPC42-mCherry (T8784 [iv]) were observed in asynchronous culture. Cells in telophase–G1 phase were selected. The scale bar represents 1 μm.

(B) Cdc7 and Sld7 colocalize with centromeres when they are detached from a spindle pole. T8888 and T8886 cells (same as T10876 and T10875 but with mad2Δ bub2Δ) were observed as in Figure 1C.

(C) Sld7 localization at centromeric regions is dependent on pre-RC. CDC6+ (wild-type [WT], T9126) and GAL-CDC6 (T9127) cells with SLD7-GFP SPC42-CFP (i), as well as ORC2+ (WT, T9273) and GAL-orc2-1 (T9272) cells with SLD7-GFP SPC42-mCherry NIC96-mCherry rad9Δ rad24Δ mad2Δ (ii), were treated with α factor, released from it, and treated with α factor again. GAL promoter (GALp) was shut off during this process (see Supplemental Experimental Procedures). Cells were observed after cytokinesis.

(D) Cdc7 localization on centromeric regions is independent of pre-RC. GAL-CDC6 cells (T9125) with CDC7-GFP SPC42-CFP NIC96-mCherry (i), as well as ORC2+ (WT, T9275) and GAL-orc2-1 (T9274) cells with CDC7-GFP SPC42-mCherry NIC96-mCherry rad9Δ rad24Δ mad2Δ (ii), were treated with α factor, released from it, and observed in 120 min. GALp was active (ON) or shut off (OFF).

(E) Cdc7 and its kinase activity are required for Sld7 localization on centromeric regions. (i) CDC7+ (WT, T8613) and cdc7-4 (T9085) cells with SLD7-GFP SPC42-mCherry were incubated at 25°C for 2.5 hr and then at 37°C for 35 min, in the presence of α factor. (ii) CDC7+ (WT, T8613) and cdc7-as3 (T10417) cells with SLD7-GFP SPC42-mCherry were arrested in G1 phase with α factor and then treated with 20 μM PP1 (inhibitor) for 20 min in the presence of α factor.

(F) Summary of results in Figure 2. See details in the text.

See also Figure S1.