Article Figures & Data
Figures
- Fig. 1.
Chromatin binding of Rad17 is enhanced following DNA damage. (A) Schematic outline of the chromatin fractionation assay. Details of the assay are described in Materials and Methods. Histone H4 and α-tubulin were used as controls for chromatin-bound and non-chromatin-bound proteins in either hydroxyurea- or MMS-treated or -untreated cells with anti-acetyl-histone H4 (Lys 16) polyclonal antibody (Upstate Biotechnology) or antitubulin monoclonal antibody, respectively. HU, hydroxyurea. (B) Chromatin association of Rad17 and Rfc2 following MMS treatment. Cells were grown in 0.05% MMS. At the indicated times, cells were fractionated as outlined for panel A and as described in Materials and Methods. Spheroplasts were prepared from cells containing rad17 +:myc(left panel) or rfc2 +:HA(right panel). Twenty micrograms of total protein, an equivalent volume of the supernatant (Sup), and five volume equivalents of chromatin-bound proteins (Chr) were fractionated on an 8% SDS gel and analyzed by Western blotting with anti-myc monoclonal antibody (9E10). (C) Chromatin association of Rad17 after ionizing radiation. Cells containing rad17:myc were irradiated with 250 Gy and recovered by incubation at 25°C for 20 min and fractionated as described for panel A. (D) Total cell extracts were prepared under denaturing conditions from wild-type (untaggedrad17 +) and myc-taggedrad17 + cells and were probed with anti-myc antibody. The arrows mark the two forms of Rad17:myc.
- Fig. 2.
The chromatin binding of Rad17 in response to S-phase perturbation. (A) The mid-log-phaserad17 +:myc strain was incubated in medium containing 12 mM hydroxyurea. At the indicated times, cell samples were removed for FACS analysis and chromatin fractionation assay by probing with anti-myc monoclonal antibody. (B) Rfc1 and Rfc2 were chromatin bound in cells grown in hydroxyurea. Mid-log-phase cells expressed Rfc1:HA in arad17 + background, or Rfc2:HA in arad17 + or rad17Δ background was incubated in medium with 12 mM hydroxyurea for 3 h. Chromatin fractionation assay was performed and analyzed by Western blotting and was probed with anti-HA monoclonal antibody. (C) Mid-log-phase cells ofrad17 +:myc in acdc22-M45 background grown at 25°C were shifted to 36°C for 3 h. Cell samples grown at 25 and 36°C were removed for chromatin fractionation assay. (D) Mid-log-phase cells that expressed Rad17:myc from genomic loci in cdc20 andpolαts13 backgrounds grown at 25°C were shifted to 36°C for 2 h. Chromatin fractionation was performed, analyzed by Western blotting, and probed with anti-myc monoclonal antibody. (E) Mid-log-phase cells that expressed Rad17:myc from genomic loci incds1Δ and rad3Δ backgrounds were incubated in media containing 12 mM hydroxyurea. Chromatin fractionation assay was performed and analyzed by Western blotting and was probed with anti-myc monoclonal antibody.
- Fig. 3.
The checkpoint-defective Rad17(k118E) mutant protein localizes in the nucleus but has reduced ability to bind chromatin. (A) Logarithmically growing rad17Δ cells harboring pREP41-myc-rad17 + or pREP41-myc-rad17.K118E and wild-type untaggedrad17 + cells were harvested and processed for immunofluorescence analysis as described in Materials and Methods. (B) Chromatin fractionation assay was performed with logarithmically growing rad17Δ cells harboring pREP41-myc-rad17 + or pREP41-myc-rad17.K118E as described in Materials and Methods. (C) Cds1 kinase activities of therad17 + wild type and mutantrad17.K118E in the presence of 12 mM hydroxyurea. The indicated strains were grown at 25°C in either the absence or presence of 12 mM hydroxyurea for 3 h. Cds1 was immunoprecipitated from 1 mg of soluble protein and assayed for kinase activity as described in Materials and Methods (upper panel, 32P). The relative amounts of Cds1 used in the kinase assay were estimated by Western blotting (lower panel, anti-myc).
- Fig. 4.
Purified Rad17 protein, but not the checkpoint-defective Rad17(K118E) mutant protein, binds ATP in vitro. (A) Silver-stained polyacrylamide gel of 0.2 μg of purified wild-type Rad17 and mutant Rad17(K118E) proteins. (B) Wild-type Rad17, but not mutant Rad17(K118E), binds ATP. [γ-32P]ATP was incubated with purified Rad17 (left panel) or purified mutant Rad17(K118E) protein (right panel) in the presence or absence of 96-mer oligonucleotide or M13 DNA and was processed as described in Materials and Methods. The bottom panel shows the Western blot analysis of the purified Rad17 and Rad17(K118E) proteins used for the ATP binding assay.
- Fig. 5.
Rad17 from a checkpoint-defective mutant,rad17.K118E, has reduced ability to associate with Rfc2. (A) Wild-type Rad17 can complex with Rfc2 but not with Rfc1. Crude cell lysates were prepared from 5 × 108 rad17Δ cells expressing either the HA-taggedrfc1 + or HA-tagged rfc2 +at their endogenous chromosomal loci and myc-Rad17 from pREP41-myc-rad17 +. Cell lysates (300 μl) containing 1 mg of protein were used for immunoprecipitation (IP) by anti-HA antibody. The immunoprecipitates were Western blotted with either anti-HA (right panel) or anti-myc monoclonal antibody (left panel). (B) Mutant Rad17(K118E) from a checkpoint-defective strain has significantly reduced ability to associate with Rfc2. Lysates from 5 × 108 rad17Δ cells containing HA-tagged rfc2 + at its endogenous chromosomal locus and pREP41-myc-rad(K118E) were prepared and immunoprecipitated with anti-HA monoclonal antibody, and the immunoprecipitates were analyzed as for panel A. The cell lysate panels show that all strains have adequate expression of the epitope-tagged proteins. (C) Association of wild-type Rad17 and mutant Rad17(K118E) proteins with Rfc2 following MMS treatment. Strains described for panels A and B were grown in 0.05% MMS for 3 h. Cells (5 × 108) were removed for immunoprecipitation with anti-HA monoclonal antibody and were probed with anti-myc monoclonal antibody. (D) Association of wild-type Rad17 and mutant Rad17(K118E) proteins with Rfc2:HA following hydroxyurea (HU) treatment. Cells (5 × 108) from strains described for panels A and B were grown in media containing 12 mM hydroxyurea for 3 h. Cell lysates were prepared and immunoprecipitated with anti-HA monoclonal antibody and Western blotted with anti-myc monoclonal antibody.
- Fig. 6.
Cells with deletion of rfc1 are checkpoint proficient. (A) Restriction map and open reading frame (ORF) ofrfc1 +. A 1.8-kb ura4 +gene cassette was used to replace 1.7 kb of theEcoRI-PstI coding region as described in Materials and Methods. (B) rfc1 + is an essential gene. Spores derived from the heterozygous diploidrfc1 +/rfc1Δ were analyzed by tetrad dissection after incubation on yeast extract agar plates at 30°C for 4 days. The 2:2 segregation of the germinating spores indicates thatrfc1 + is essential for cell viability. (C) Germinating rfc1Δ spores have elongated cell morphology. Shown are cells derived from a single germinating spore. (D) Cells withrfc1Δ have intact checkpoint processes. DAPI staining of germinating rfc1Δ spores having elongated cell morphology and normal nuclear morphology is shown. (E) FACS profiles. Heterozygous diploid rfc1 +/rfc1Δ and controlura4 +/ura4D-18 diploid cells were germinated at 30°C in minimal media lacking uracil. Germinating spores were collected every 2 h, fixed with 70% ethanol, and analyzed by FACS as described in Materials and Methods. The positions of 1C and 2C DNA peaks are indicated.
- Fig. 7.
A proposed model illustrating how Rad17 binds to chromatin in response to aberrant genomic structures induced by damage, replication mutant arrest, and hydroxyurea block in the presence or absence of an activated Cds1 kinase. P, phosphorylation.
