PMC

Separation of function analysis of the “intra-S checkpoint”. A) Flow cytometry of cells blocked in G1 phase with alpha factor and then either released into nocodazole (dotted lines) or into 0.033% MMS (grey). B) Rad53 western blot from experiment in A). C) Viability of cells plated from experiment in A, error bars are standard error of the mean, n=4. D) Model of inhibition of CDK and DDK in G1 phase by the APC/C or in S-phase by Rad53.

Sld3 and Dbf4 are the minimal substrates of Rad53 for the block to origin firing. A) Top - Southern blot of alkaline gel of replication intermediates from cells arrested in G1 with alpha factor and released into HU for the indicated times and probed for specific origins as indicated. –B) Rad53 western blot of experiment in a). C) Western blot of Dbf4 (top) and Rad53 (bottom) after G1 arrest with alpha factor (α) and release into 200mM HU for the indicated times. The rad53Δ strain is also sml1Δ. * indicates a non-specific band. D) as in A) bottom – Rad53 western blot. E) 2D neutral/neutral electrophoresis of replication intermediates from cells arrested for 90 mins in HU after release from G1 phase. The Msc1 digest was probed first for ARS501 and then ARS315. F) as in A). For lanes 1-12 the allele dbf4-19A, sld3-38A or both are present as second copies in haploid yeast.

At least two essential functions of Sld3 are inhibited by Rad53. A) Sld3 C-terminus (530-668), which was first phosphorylated with hot ATP and CDK, was incubated without (−) or with increasing amounts of Rad53 and cold ATP (+). This protein was then used in a pulldown with Dpb11-GST (1-395) (WT) or (90-395) (ΔN). Top -autoradiogram, bottom – coomassie. B) Dpb11-GST pulldown assay as in A, with CDK phosphorylated full length Sld3 either with all the alanine mutations (38A) or with different subsets of the aspartic acid mutations (12D, 14D and 20D). C) Viability and tetrad analysis of diploids heterozygous for the corresponding Sld3 allele. Sld3 is full length, Dpb11 fusion is 253-764. D) Yeast-two-hybrid assays between the indicated baits and full length Sld3, either wild type or with S306D, T310D mutations (2D). E) Tetrad analysis on YP-galactose of a diploid heterozygous for sld3-14D-DPB11 fusion and Gal-CDC45. Circles are the sld3-14D-DPB11 fusion allele alone. Squares are the sld3-14D-DPB11 fusion allele + Gal-CDC45.

Sld3 is novel target of Rad53 in vivo. A) Western blot after release of alpha factor arrested cells (G1) into 200mM HU. B) Anti-myc western blot of purified His/Myc-tagged Sld3 from yeast cells arrested as in A). C) schematic representation of yeast Sld3. All serine/threonine residues are marked by black bars (top), with the position of the identified Rad53 sites in either the Sld3 alanine mutant (open circles) or aspartic acid mutants (closed circles) below. The essential CDK sites (600/622) and the position of the TEV protease site for figure 1e are shown above. The position of the 5 Sld3 fragments in Figure 1d are shown below. D) in vitro Rad53 kinase assay with bacterially expressed Sld3 fragments 1-5. E) Western blots of purified and cleaved Sld3-TEV allele from HU arrested cells. This allele contains a myc tag at the C-terminus, HA-tag in the middle, with a TEV protease cleavage site in-between. This allele is viable as the only copy in yeast. F) Western blot of Sld3-13myc from cells arrested in G1 with alpha factor and released into HU for the indicated times.