Fig 3.

Alanine substitution mutations in the inter-BRCT region of Dpb11 cause defects in DNA replication. (A) Amino acid sequences of aa 253 to 291 of Dpb11. The mutation sites in each mutant are shown. (B) The yeast two-hybrid assay was performed as described for Fig. 2B. (C) Low-copy-number dpb11-AAA8/9/12/13 was introduced into YST756 (Δdpb11 [YEp-DPB11]) and grown on a 5-FOA-containing plate to eliminate the YEp-DPB11 plasmid from the cells. The cells grown on 5-FOA were serially diluted and grown on YPAD at the respective temperatures indicated. (D) Low-copy-number DPB11 constructs (wild-type DPB11 [Wt] and AAA mutants [AAA6 to AAA8/9/12/13]) were introduced into YST756 (Δdpb11 [YEp-DPB11]), and cells that lost YEp-DPB11 were recovered as described for panel C. These cells were serially diluted and grown on YPAD or YPAD containing 150 mM HU at 30°C. (E) YST1520 (GALp vector), YST1523 [GALp-SD fusion (253)], and YST1969 [GALp-SD fusion (253-AAAs)] cells were grown and analyzed as described for Fig. 1B. (F) Cells with wild-type DPB11 (W303-1a Δbar1 [Wt]), dpb11-AAA6 (YST1977 and YST1978 [AAA6]), and dpb11-AAA8/9/12/13 (YST1987, YST1988 and, YST1989 [AAA8/9/12/13]) were serially diluted and grown on YPAD or YPAD containing 100 mM HU at 30°C. (G) W303-1a Δbar1 [DPB11 (Wt)] and YST1987 (dpb11-AAA8/9/12/13) cells were grown in YPAD medium at 30°C (Asyn.), arrested in the G₁ phase using the alpha factor, and synchronously released at 30°C. Samples were taken at every 10 min after the release (0 to 90 min). The DNA content of the samples was analyzed by flow cytometry. Flow cytometry profiles of samples under conditions of DNA replication judged from the peak position are filled in yellow. (H) The experiment was performed as described for panel G, with the exception that cells were grown at 25°C. (I and J) The proportions of budded cells of the same samples as those described for panels G and H, respectively, are shown.