Figure 5.

Involvement of septin Shs1 in the response to DNA replication stress. (A) Deletion of SHS1 rescues the elongated bud phenotype induced by FHA1 domain overexpression. Protein extract of the cell cultures were prepared 10 h after galactose induction, and the expression level of the FHA domains was monitored by anti-Xpress Western blot. The membrane was stained with Ponceau for loading control. (B) shs1Δ cells exhibit elongated buds after treatment with 150 mM HU for 6 h. (C) In vitro phosphorylation of individual septin components by Rad53. Each septin was expressed and purified from E. coli. Among these septins, Shs1 is the only septin efficiently phosphorylated by Rad53. (D) shs1Δ cells are hypersensitive to replication stress at 37°C. Indicated cells (in RDKY5763 background) were plated on normal YPD plates or freshly prepared YPD plates containing 75 mM HU or 0.05% MMS. Cells were then grown at 30 or 37°C. Shs1 is required for cell viability at 37°C in the presence of HU. Note that shs1Δ cells are considerably more sensitive than dun1Δ cells under HU at 37°C. A serial dilution of threefold was used.