Analysis of Rad53 FHA1 interactions with Cdc7 and Cdc45 in the GST pull-down assay. A, Total extracts (0.3 mg protein) from asynchronous cultures of yeast cells containing a TAP-tagged version of Cdc7 or Cdc45 (MCM974 and MCM973, respectively) were incubated with equal amounts (150 μg) of immobilized GST or GST-FHA1. Bound proteins were analyzed by Western blotting using anti-TAP antibody. B and C, Yeast cells containing a TAP-tagged version of Cdc7 or Cdc45 (MCM974 and MCM973, respectively) were synchronized in G1 with α-factor (G1), released into S phase and either left untreated for 30 min (S) or treated either with camptothecin (20 μg/ml) for 1 h (S + CPT), or with hydroxyurea (200 mm) for 2 h (S + HU). Total extracts were incubated with immobilized GST or GST-FHA1, and bound proteins were analyzed by Western blotting as above.

A, B, The interactions between FHA1 [Rad53(1–164)] and either Cdc7(294–493) (A) or Cdc45(154–270) (B) depend upon T484 and T189, respectively, in the two-hybrid assay. pGBT9/FHA1 or pGBT9/FHA1R70A expressing the Gal4BD-Rad53(1–164) (wt) and Gal4BD-Rad53(1–164)R70A (m) fusion proteins, respectively, were introduced into the tester strain along with wild-type or mutated pACTII/Cdc7(294–493) or pACTII/Cdc45(154–270) vectors harboring the sequence encoding Gal4AD fused to the sequences encoding wild-type (WT) Cdc7(294–493) or Cdc45(154–270), or their mutated derivatives as indicated. - indicates that empty vectors (either pGBT9 or pACTII) were used as controls. The two-hybrid interactions were revealed by growth on plates lacking histidine (-His) complemented with 100 mm 3-amino-triazole (3AT) and by X-gal staining. Control plates contained 3AT but were complemented with histidine (+His). It has to be noted that the Gal4BD-Rad53(1–164) fusion protein can activate by itself the transcription of the reporter genes at a low level, hence the residual growth and the slight blue coloration visible on the first spot of the plates (for transformants containing pGBT9/FHA1 and the empty vector pACTII). C and D, Cdc7 and Cdc45 interactions with Rad53 FHA1 are dependent upon T484 and T189, respectively, in the GST pull-down assay. Total extracts (0.3 mg protein) from asynchronous cultures of yeast cells containing a TAP-tagged version of either the wild-type Cdc7 or Cdc45 (MCM969 and MCM965, respectively) or the mutated Cdc7T484A or Cdc45T189A (MCM971 and MCM967, respectively) proteins were incubated with immobilized GST or GST-FHA1. Bound proteins were analyzed by Western blotting as above. E, Co-immunoprecipitation analysis of Cdc7 and Cdc45 interactions with Rad53. The pJA98Ade plasmid harboring RAD53 under the control of a GAL promoter was introduced into the control strain MCM185 (Control) and into the strains MCM969, MCM971, MCM965, MCM967 containing the CDC7-TAP, cdc7T484A-TAP, CDC45-TAP and cdc45T189A-TAP constructs, respectively. Cells were grown overnight in minimal media containing raffinose and expression of Rad53 from the pJA98Ade plasmid was induced by addition of galactose. The TAP-tagged proteins were immunoprecipitated using PAP antibodies. Bound proteins were analyzed by Western blotting using an antibody directed against Rad53 that also cross-reacted with the TAP-tagged proteins. 10% of the extract (input) was analyzed using antibodies directed against the C terminus sequence of Rad53p.

Screen capture of the STRIP web server with the query page on the left and the result page on the right. A dark blue color indicates that the corresponding phosphoresidue satisfies either the phosphorylability, the consensus motif rule or the strict conservation condition. Light blue cells indicate that the conservation condition is only respected with the less stringent condition and in that case, further manual analysis may be performed easily. By clicking in the table cell of any phosphoresidue, a pop-up window allows for rapidly checking the conservation features between S. cerevisiae close homologs, detecting possible sequence truncations or identifying alignment flaws.

A, Cdc7 threonine STRIP analysis. B, Cdc45 threonine STRIP analysis. Threonines in bold and italics correspond to those present inside and outside the interacting fragments identified by the two-hybrid screens, respectively.

Graph summarizing the proteins found to physically interact with Rad53 in affinity-based (blue links) and two-hybrid (red links) experiments as collected in the Biogrid database. Color codes refer to the Gene Ontology definitions used in the Osprey visualization program. Cdc7 and Cdc45 gene names are colored red and the other gene names that were studied in this work are labeled with an obelisk (‡). Proteins found to specifically interact with the isolated FHA1 domain of Rad53 through affinity-based experiments are labeled with an asterisk (*) (18). When possible, gene names were clustered with respect to their connectivity and to their biological function in black boxes. Plain or dashed underlines indicate proteins bearing a putative FHA1 binding phosphothreonine as detected using the stringent or the more permissive predictive criteria, respectively.

Screen capture of the STRIP web server with the prediction of likely kinases according to five different methods, namely NetPhosK (34), PredPhospho (35), PPSP (36), ScanSite (37) and KinasePhos (38). In the main page the class name of the predicted kinase together with the number of servers that converged on the same prediction is provided. By clicking in the table cell of this consensus prediction, a pop-up window provides a detailed analysis of the predictions. In addition, all the S. cerevisiae kinases classified in the same class as the predicted kinase (82) are mentioned in the tables below.