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First published online 6 January 2004
doi: 10.1242/jcs.00907

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A domain of Rad9 specifically required for activation of Chk1 in budding yeast

Richard T. Blankley and David Lydall^*,

School of Biological Sciences, University of Manchester, G38 Stopford Building, Oxford Rd, Manchester, M13 9PT, UK

View larger version (37K): [in a new window]   Fig. 1. Genetic separation of function screen. (A) The `checkpoint arrest' assay measures colony growth at the semi-permissive temperature of 27.3°C. In this assay, RAD9 cdc13-1 cells do not form colonies because they enter a G2/M arrest. By contrast, rad9 cdc13-1 cells cannot arrest and do form colonies. The rapid death assay measures colony growth after three periods of growth at the restrictive temperature of 36°C for 4 hours and finally a return to growth at 23°C. In this assay RAD9 cdc13-1 cells retain viability and form colonies, whereas rad9 cdc13-1 cells lose viability in this assay. In practice, to distinguish better between the growth of RAD9+ and rad9 cells, plates were subjected to three 4-hour periods at the restrictive temperature of 36°C, separated by 4-hour periods of recovery at the permissive temperature of 23°C. Colonies were then allowed to form at 23°C. (B) Novel alleles rad9i-1 and rad9s-2 have opposite phenotypes in the two assays. pRS416 plasmids containing the RAD9 (lanes 1,2), rad9i-1 (lanes 5,6) and rad9s-2 (lanes 7,8) alleles were transformed into a rad9 cdc13-1 strain. An empty vector was transformed into lanes 3 and 4. A 1:5 dilution series was prepared and spotted onto SC-uracil (left two) or YEPD (right) plates, which were incubated as shown. (C) Wild-type RAD9 is 1309 amino acids, contains an [S/T]Q cluster domain (SCD) and two BRCT domains. (D) The rad9i-1 allele contains an in-frame 5 amino acid insertion of GMFKH after C853. (E) The rad9s-2 allele contains a transposon insertion into the 5' half of the RAD9 gene. The first 711 bp of the RAD9 coding sequence have been removed and replaced by 335 bp of transposon sequence (shown in black). The longest open reading frame remaining encodes an N-terminal truncation of Rad9 using the ATG at methionine M243 as a start codon.

View larger version (76K): [in a new window]   Fig. 2. Deletion analysis of the Rad9 N-terminus. (A) Phenotypes of truncation and internal deletion RAD9 alleles in two growth assays. A series of N-terminal truncations and internal deletions on single copy plasmids transformed into a rad9 cdc13-1 strain. The gaps in the black bars represent in-frame deletions of the coding sequence. Numbers over the black bars correspond to the amino acids that remain flanking the deletions. Data is not shown for constructs rad91-159, rad91-199, rad91-239, rad940-159, rad980-119, rad980-159, rad980-231 or rad9120-159, all of which had intermediate-strength rad9s phenotypes similar to those shown in rows 4-6 and 8-10. (B) The rad91-231 allele has the strongest rad9s phenotype when integrated; de novo deletion alleles of RAD9 were integrated at the URA3 locus of a rad9::LEU2 cdc13-1 cdc15-2 strain and tested in the two growth assays.

View larger version (30K): [in a new window]   Fig. 3. N-terminal domains of Rad9 are comparatively well conserved in other yeast species. Rad9 orthologues in two divergent budding yeasts (Saccharomyces castelli and Saccharomyces kluyveri) were obtained from the Saccharomyces Genome Database. Amino acids 40-200 of the three Rad9 orthologues were aligned with amino acids 40-200 from S. pombe Crb2 using the ClustalW algorithm. Perfectly conserved residues are highlighted in black, structurally similar residues are highlighted in grey.

View larger version (59K): [in a new window]   Fig. 4. rad91-231 mutants have similar phenotypes to chk1 mutants. Strains of the indicated genotypes were grown in liquid culture before a fivefold dilution series was prepared and spotted to YEPD plates. Strain numbers are in parenthesis. (A) rad91-231 mutants are not UV sensitive. Plates were untreated or exposed to 40 J/m2 UV before incubation for three days at 30°C. (B) yku70 rad91-231 cells are checkpoint arrest defective at restrictive temperatures. Growth was measured at the permissive temperature of 30°C and the restrictive temperature of 37°C. (C) Arrest kinetics of cdc13-1 mutants in synchronous cultures. Checkpoint mutations were combined with the cdc13-1 and cdc15-2 mutations. Cultures were arrested with -factor at 23°C then released into fresh media at 36°C. Accumulation of cells at mid-nuclear division (cdc13-1 arrest point) and late nuclear division (cdc15-2 arrest point) was measured by fluorescence microscopy at intervals of 20 minutes.

View larger version (47K): [in a new window]   Fig. 5. MMS-induced Chk1 phosphorylation is abrogated in rad91-231 mutants. Indicated strains containing the cdc15-2 allele were transformed with a CHK1-HA plasmid and cultured in selective media. Cultures were arrested in telophase by incubation for 3 hours at 37°C before being mock treated or treated with 0.1% MMS (v/v) for 1 hour. Whole cell extracts were prepared and immunoblotted with -Rad9, -Rad53 or -HA antibodies. The same phosphorylation patterns were observed with two separate extracts and Chk1 phosphorylation was detected with both HA- and Myc-tagged proteins.