Article Figures & Data
Figures
- FIG. 1.
The nmt41xmcl1-MH mutant is phenotypically similar to the mcl1-1 mutant. (A) Growth of the nmt41xmcl1-MH55 strain and the parental mcl1+ strain at 25°C for 25 generations in selective medium containing no thiamine (25/25/on), with 10 mM thiamine to repress nmt gene expression (25/25/off), or at 36°C (36/25/off). The lower panel is a phase-contrast light microscope image of nmt41xmcl1-MH55 cells from the liquid culture prior to plating (bar in phase-contrast images represent 5 μm). (B) Recovered double mutant nmt41xmcl1-MH bub1Δ and nmt41xmcl1-MH mad2Δ strains were struck onto selective media containing 0 (on) or 10 (off) mM thiamine to examine the differences in the genetic interactions between nmt41xmcl1-MH and the two spindle checkpoint mutations at permissive temperature of 25°C. (C) Cultures of nmt41xmcl1-MH, cds1Δnmt41xmcl1-MH (603), cds1Δ (FY865), rad3Δ (591), mcl1-1 (546), and mcl1-GFP (551) cells were grown for 25 generations in selective media and then split into medium without (on) or with (off) 10 mM thiamine for 6 generations before plating on YES agar plates (a thiamine-enriched medium) containing 0.0025% MMS, 5 mM HU, or 10 mg of TBZ/ml. Cells were serially diluted 1:5 from 106 to 102 cells/ml.
- FIG. 2.
Mcl1p interacts with N-terminal fragments of Pol1p. (A) Schematic representation of the two epitope-tagged alleles of mcl1 (strains 551 and 589) used in this study. (B) Schematic representation of the polymerase α holoenzyme showing the three conserved domains of the eukaryotic Pol1 (p180): an acidic N terminus, a central deoxynucleotide transferase catalytic core composed of seven B-polymerase as well as five polymerase α conserved sequences, and the C-terminal Zn finger domain with B subunit and primase association presented. In the lower panel, the two bacterially expressed GST-Pol1p fragments used for in vitro interaction studies presented in panel C and a fission yeast-expressed Pol1-GFP protein fragment used for in vivo interaction studies presented in panels D and E are shown. (C) Western blot analysis of GST pulldowns from yeast lysates containing Mcl1-GFP (left) or Mcl1-MHp (center) with the various GST proteins (right). The epitope-tagged Mcl1 proteins strongly interact with the GST-NT fragment (center lanes) but not the GST or GST-CT proteins (right and left lanes). (D) Mcl1-MHp interacts in vivo with a small fragment of Pol1p tagged with GFP (GFP-NT). Yeasts were grown in conditions where the nmt promoter of mcl1-MH was either induced (ON) or repressed with thiamine (OFF). The six-His-tagged Mcl1-MHp was recovered with cobalt-immobolized agarose resin (Talon). Mcl1-MHp interaction with GFP-NT was not disrupted by DNase I digestion of Talon-bound material. Additionally, no interaction was seen between Mcl1-MH and GFP alone. (E) Cell cycle restriction points for the temperature-sensitive cdc25-22 (G2 arrest of strain 596), orp1-4 (pre-S arrest of strain 599), cdc22-M45 (early S arrest of strain 597), or cdc21-M68 (mid-S arrest of strain 598) mutants were used to test for a cell cycle-dependent interaction between Mcl1-MHp and GFP-NT. Only those cells arrested in S phase (flow cytometry not shown) had enrichment of GFP-NT in the Mcl1-MHp pulldown with Talon resin.
- FIG. 3.
mcl1-1 mutants are sensitive to DNA damage specifically in S phase. (A) Cultures of cdc10-129 cells (strain 53) were arrested for 3 h at 36°C and then released into either YES, YES with 12 mM HU, or YES with 0.02% MMS. Cells were collected to determine DNA content by flow cytometry (top row of three panels) or relative viability, as determined by serial dilution plating (middle row of panels). Cells plated from untreated cultures were also irradiated with 100 J of UV light/m2 at given time points (bottom panels). (B and C) cdc10-129 rad3Δ cells (strain 591) and cdc10-129 mcl1-1 cells (strain 578), respectively, were treated as described above. Data presented is representative of four independent experiments.
- FIG. 4.
DNA replication forks at ars2-1 in mcl1-1 mutants examined during replication stress are stable and persistent. The three cdc10-129 strains shown in Fig. 3 were arrested at 36°C and released into 5 mM HU to induce replication stress. Genotypes of strains are given at the top of each column, and each row is labeled from the corresponding time points. (A) Flow cytometric analysis of DNA content in cells released into 5 mM HU. Cytology taken through the experiment showed that most cells entered mitosis by 240 min, except for mcl1-1 cells, which appeared cell cycle arrested at this time point (data not shown). (B) Relative viability was assessed in serial plating of cultures at each time point. (C) Southern blot analysis of 10 μg of total DNA by two-dimensional gels probed for ars2-1 sequences (an early firing origin). Quantification of total signal with Molecular Dynamic ImageQuant 5.1 shows that the hybridization signal increases twofoldin the cdc10-129 (3 × 105 to 6 × 105 counts) and rad3Δ (3 × 105 to 5.5 × 105 counts) strains but not the mcl1-1 strain (2 × 105 to 2.4 × 105 counts). Filled arrowheads mark the bubble arc, and open arrowheads mark the X line. (D) Diagrammatic representation of two-dimensional DNA gel patterns showing how autonomously replicating sequence DNA structure relates to mobility in two-dimensional gel electrophoresis, which is adapted from http://fangman-brewer.genetics.washington.edu/2Dgel.html .
- FIG. 5.
Hsk1 and Cds1 affect the localization of Mcl1p to chromatin during HU arrest. (A) The percentage of cells that retained Mcl1-GFP in association with chromatin following chromatin extraction procedures is plotted in the histogram, with error bars representing standard error of the mean from four experiments. Cells were at 36°C for 3 h, and cell cycle arrests were confirmed by flow cytometric quantification of DNA content for each experiment (data not shown). (B, C, and D) Representative deconvolved fluorescence images of mcl1-GFP (strain 551; panel B), cds1Δ mcl1-GFP (strain 553; panel C), and hsk1-1312 mcl1-GFP (strain 555; panel D) cells are shown after chromatin extraction. DNA stained with Hoecsht 33258 is false colored red, Mcl1-GFP is in green, and colocalized signal appears yellow. (E) To quantify the remaining Mcl1-GFP in the chromatin fraction, the postextraction chromatin-containing material was separated by sodium dodecyl sulfate-8% polyacrylamide gel electrophoresis and transferred by Western blotting for immunodetection of Mcl1-GFP: lane 1, mcl1-GFP; lane 2, cds1Δ; and lane 3, hsk1-1312. The bottom panel is Coomassie-stained gel loaded identically for a loading control, since protein expression appears altered in the hsk1 mutant background.
- FIG. 6.
Checkpoint kinases affect Mcl1p complexes. (A) Sucrose gradient sedimentation of Mcl1-GFP from nonmutant (mcl1-GFP), cds1Δ, rad3Δ, and hsk1-1312 cells arrested with 12 mM HU and cellswhere Cds1p was overexpressed (nmt1GST-cds1) prior to 12 mM HU arrest. Gradients were fractioned into 22 0.5-ml fractions from the bottom (fraction 1) of the gradient. For reference, sedimentation of known standards with their relative molecular weights and sedimentation values are shown at the bottom of the α-GFP Western blots. (B) Western blot analysis of α-GFP immunoprecipitations from sucrose gradient fractions containing Mcl1-GFP show altered association with Pol1p in hsk1-1312 and nmt1GST-cds1 backgrounds but not rad3Δ and cds1Δ backgrounds. (C) GST-Pol1118-634-GSH-Sepharose coprecipitates Pol1p (top panel) and Mcl1GFP (middle panel) from mcl1-GFP, hsk1-1312, and cds1Δ but not from nmtGST-cds1 whole-cell extracts. Immunodetection of GST molecules shows that this result is not due to the displacement of the GST-Pol1118-634 from the GSH-Sepharose by the coexpression of GST-Cds1p.
Tables
- TABLE 1.
Yeast strains
Strain Source Genotype 53 cdc10-129 leu1-32 99 h − ade6-M210 leu1-32 ura4-D18 his3-D1 546 h − mcl1-1 ade6-M210 leu1-32 ura4-D18 his3-D1 547 S. Sazar h − mad2Δ::ura4+ade6-M216 leu1-32 ura4-D18 549 J. P. Javarzet h − bub1Δ::ura4+ade6-M216 leu1-32 ura4-D18 551 h − mcl1GFP::ura4+ade6-M210 leu1-32 ura4-D18 his3-D1 553 h − mcl1GFP::ura4+cds1Δ::ura4+ade6-M210 leu1-32 ura4-D18 his3-D1 554 h + mcl1GFP::ura4+nmt1::GSTcds1 LEU2 ade6-704 leu1-32 ura4-D18 555 h − mcl1GFP::ura4+hsk1-1312 ade6-M210 leu1-32 ura4-D18 his3-D1 558 h − mcl1GFP::ura4+cdc25-22 ade6-M210 leu1-32 ura4-D18 his3-D1 561 h − mcl1GFP::ura4+cdc22-M45 ade6-M210 leu1-32 ura4-D18 his3-D1 560 h − mcl1GFP::ura4+cdc10-129 ade6-M210 leu1-32 ura4-D18 his3-D1 562 h − mcl1GFP::ura4+rad3Δ::ura4+ade6-M210 leu1-32 ura4-D18 his3-D1 578 h − mcl1-1 cdc10-129 leu1-32 590 G. Freyer h − rqh1Δ::ura4+ade6-M210 lueu1-32 ura4-D18 591 h − rad3Δ::ura4+cdc10-129 leu1-32 592 h − nmt41x-mcl1-MH::ura4+ade6-M210 leu1-32 ura4-D18 his3-D1 593 h − nmt41x-mcl1-MH::ura4+ade6-M210 leu1-32 ura4-D18 his3-D1 cdc25-22 594 h − nmt41x-mcl1-MH::ura4+ade6-M210 leu1-32 ura4-D18 his3-D1 cdc22-M45 595 h − nmt41x-mcl1-MH::ura4+ade6-M210 leu1-32 ura4-D18 his3-D1 cdc21-M68 596 h − nmt41x-mcl1-MH::ura4+ade6-M210 leu1-32 ura4-D18 his3-D1 cdc10-129 597 h − nmt41x-mcl1-MH::ura4+ade6-M210 leu1-32 ura4-D18 his3-D1 orp1-4 598 h − nmt41x-mcl1-MH::ura4+ade6-M210 leu1-32 ura4-D18 his3-D1/pTB19 599 h − nmt41x-mcl1-MH::ura4+ade6-M210 leu1-32 ura4-D18 his3-D1 cdc25-22/pTB19 600 h − nmt41x-mcl1-MH::ura4+ade6-M210 leu1-32 ura4-D18 his3-D1 cdc22-M45/pTB19 601 h − nmt41x-mcl1-MH::ura4+ade6-M210 leu1-32 ura4-D18 his3-D1 cdc21-M68/pTB19 602 h − nmt41x-mcl1-MH::ura4+ade6-M210 leu1-32 ura4-D18 his3-D1 orp1-4/pTB19 603 h − nmt41x-mcl1-MH::ura4+ade6-M210 leu1-32 ura4-D18 his3-D1 cd1Δ::ura4+/pTB19 1123 A. Carr h − rad26Δ::ura4+ade6-704 leu1-32 ura4-D18 FY865 S. L. Forsburg h − cds1Δ::ura4+leu1-32 ura4-D18 FY945 S. L. Forsburg h − hsk1-1312 ade6-M210 leu1-32 ura4-D18 FY1105 S. L. Forsburg h + rad3Δ::ura4+ade6-M216 leu1-32 ura4-D18 - TABLE 2.
Genetic interactionsa
Mutation Function Interaction with mcl1-1 mcl1-GFP nmt41mcl1-MH On Off mad2Δ Spindle assembly checkpoint NI NI NI NI bub1Δ Spindle assembly checkpoint kinase SL NI S SL rad3Δ DNA damage checkpoint kinase SL NI SL SL rad26Δ Rad3 interaction protein SL NI SL SL rad9Δ DNA damage checkpoint (PCNA like) S NI NI S hsk1-1312 S-phase initiation kinase SL NI ML ML cds1Δ Transducer kinase for S-phase PR NI NI PR rqh1Δ RecQ helicase homolog SL NI SL SL pol1-1 DNA polymerase-α NI NI NI NI rad22Δ Rad52 homologue NI NI rph51Δ Rad51 homologue ML NI rph54Δ Rad54 homologue ML NI ↵ a NI, no interaction; S, synthetic; SL, synthetic lethal; PR, partial rescue; and ML, meiotic lethal.
