Volume 7, Issue 4, Pages 705-713 (April 2001) An Origin-Deficient Yeast Artificial Chromosome Triggers a Cell Cycle Checkpoint  Anja J van Brabant, Christina D Buchanan, Evonne Charboneau, Walton L Fangman, Bonita J Brewer  Molecular Cell  Volume 7, Issue 4, Pages 705-713 (April 2001) DOI: 10.1016/S1097-2765(01)00216-7

Figure 1 Structure of the Wild-Type YAC (yWSS349) and the YAC Containing the Origin-Free Region (5ORIΔ) (A) Location of mapped replication origins within the wild-type YAC (van Brabant et al., 1999). The human insert sequences of the 250 kb wild-type YAC are represented by the thick bar. The location of ARS1 on the left YAC vector arm and origins A–H within the human insert sequences are shown. The small oval represents the YAC centromere. (B) Location of mapped replication origins and sites of origin deletions within the mutated YAC, 5ORIΔ. “X” represents a site-directed mutation within the ARS consensus sequence of origin D that abolishes origin function (van Brabant et al., 1999). The triangles represent deletions of origins E–G (see Experimental Procedures). The bars above the YAC refer to regions that were tested by fork-direction gel analysis (probes 1, 2, and 3; see text). The direction of replication fork movement detected by each probe is indicated by the arrows. The filled circles below the YAC correspond to the probed sequences for the data in Figure 2 (probes 4, 5, and 6 corresponding to 187 kb, 210 kb, and 232 kb from the left end of the YAC) Molecular Cell 2001 7, 705-713DOI: (10.1016/S1097-2765(01)00216-7)

Figure 2 Replication Kinetics of Three Fragments at the Right End of the Wild-Type YAC and 5ORIΔ The curves show the kinetics of replication of EcoRI fragments containing sequences hybridizing to probe 4 (solid line, filled triangles), probe 5 (solid line, filled squares), and probe 6 (solid line, filled circles) in (A) the wild-type YAC with origins intact and (B) 5ORIΔ. Probe locations are shown in Figure 1B. Minutes after release from the G1 arrest are shown on the x axis. Percent replication is plotted on the y axis and is determined from the percentages of heavy-heavy DNA and heavy-light DNA in a density-transfer experiment (McCarroll and Fangman, 1988). The early marker fragment (dashed line, open squares) contains ARS305 from yeast chromosome III (Reynolds et al., 1989), and the late marker fragment, R11 (dashed line, open circles), is located on the right end of yeast chromosome V (Ferguson and Fangman, 1992). (C) The correlation between distance from the left end of 5ORIΔ and replication time is shown. The Trep values, representing the time of half-maximal replication for each EcoRI restriction fragment, were derived from the data shown in Figure 2B. The inverse slope of the line corresponds to the rate of replication fork movement through the right end of 5ORIΔ. See Figure 1B for the location of the probed sequences relative to 5ORIΔ Molecular Cell 2001 7, 705-713DOI: (10.1016/S1097-2765(01)00216-7)

Figure 3 Doubling Time Kinetics of Individual Yeast Cells The percentage of wild-type YAC cells (circles) and 5ORIΔ cells (squares) that have failed to rebud is plotted against time in minutes following release from the α-factor block by pronase addition. The cell cycle interval from α-factor to the second bud is stylized in the inset and is not intended to represent actual cell cycle proportions Molecular Cell 2001 7, 705-713DOI: (10.1016/S1097-2765(01)00216-7)

Figure 4 Stability of 5ORIΔ Is Dependent on RAD9 (A) 5ORIΔ and the wild-type YAC were analyzed by pulsed-field gel analysis at ∼30 generations and ∼110 generations of growth under selection for uracil prototrophy. (B) Two rad9Δ spore clones containing 5ORIΔ (#1 and #2) were analyzed by pulsed-field gel analysis after ∼55, 65, and 75 generations of growth in selective medium following meiosis (lanes 2–4 and 5–7, respectively). The rad9Δ spore clone containing the wild-type YAC was similarly analyzed after ∼30 and ∼110 generations of growth in selective medium (lanes 9 and 10). The size of the full-length YACs for both 5ORIΔ and the wild-type YAC are shown in lanes 1 and 8, respectively. The pulsed-field gels were probed for URA3. A λ ladder (New England Biolabs, Beverly, MA) was run as a molecular weight standard Molecular Cell 2001 7, 705-713DOI: (10.1016/S1097-2765(01)00216-7)

Figure 5 Mapping Deletions in Variants of 5ORIΔ YAC Obtained in a rad9Δ Strain (A) 5ORIΔ YAC spore clone #2 was streaked on selective plates following 75 generations of mitotic growth in a rad9Δ background (Figure 4B, lane 7). Seven individual colonies arising on the plate were grown in a liquid medium for ∼5 generations and their DNA was isolated and analyzed by pulsed-field gel electrophoresis (lanes 2–8). The full-length 5ORIΔ YAC is shown for comparison (lane 1). (B) The shortened 5ORIΔ YACs (lanes 2–8) were analyzed by performing duplex PCR. Regions tested by PCR are indicated by black vertical bars along the length of the full-length, control YAC (gray bar, lane 1). The shaded bars indicate sequences still present in the shortened variants (lanes 2–8). The deletion end points have been placed midway between a PCR primer set that was PCR-positive and an adjacent set that was PCR-negative Molecular Cell 2001 7, 705-713DOI: (10.1016/S1097-2765(01)00216-7)

Figure 6 Rad53 Is Not Phosphorylated during the Cell Cycle in the YAC Strains Rad53 phosphorylation state was analyzed in samples from a synchronous time course experiment in the wild-type YAC and 5ORIΔ strains. Cells were synchronized in G1 (0 min.) and released into S phase. Samples were collected every 30 min and total protein extracts were analyzed for Rad53 by Western blot using an anti-Rad53 antibody. The UV lanes contain total protein extract isolated from the 5ORIΔ strain 90 min after UV-irradiation (36J/m2). The brackets indicate the migration of phosphorylated Rad53 Molecular Cell 2001 7, 705-713DOI: (10.1016/S1097-2765(01)00216-7)