Figure 1.
Construction of pYAC_MEM and YAC_MEM.
A: Name, mass and genetic map of the circular minichromosome. The relative positions of its most relevant features are indicated inside: The centromeric sequence CEN6, the autonomous replication sequence (ARS4), URA3, the lambda DNA marker sequence (L1), Tetrahymena telomeric repeats, HIS3, the lambda DNA marker sequence (L2), the ColE1 unidirectional origin (ColE1 Ori) and the ampicillin-resistance gene (AmpR). Outside, the relative positions of sites recognized by specific restriction endonucleases are indicated. B: The corresponding linear maps of the circular minichromosome’s restriction fragments and their sizes are indicated. At the bottom the genetic map of YAC_MEM. C: Circular and linear DNAs analyzed in unidirectional gel electrophoresis run in the presence of 0.1 µgr/ml chloroquine. The relative positions for linear size markers are indicated to the left. The number for each lane is shown on top and the nature of each band is shown to the right. Intact DNA isolated from S. cerevisiae transformed with pYAC_MEM (lane 1); DNA isolated from E. coli cells transformed with pYAC_MEM digested with BamHI (lane 2); Intact DNA from S. cerevisiae transformed with YAC_MEM (lane 3). Hybridized with L2. D: Genomic and extra-chromosomal DNA fragments analyzed by unidirectional gel electrophoresis after digestion with BamHI and XhoI, hybridized with URA3. DNA from untransformed cells (lane 1); DNA from cells transformed with pYAC_MEM (lane 2); DNA from cells transformed with YAC_MEM (lane 3). Note the relative intensities of the genomic (chromosomal) and extrachromosomal bands in each lane.
Figure 2.
Cell synchronization and monitor of their synchronous progression through the S-phase with and without DNA topoisomerase 2.
top2-td cells transformed with the circular minichromosome pYAC_MEM were synchronized at the G1-S boundary and released synchronously into the S-phase at either permissive or restricted conditions. Samples were taken at regular intervals, the cells stained with SYTOXGreen and analyzed by flow cytometry. The data corresponding to fluorescence-activated cell sorting analysis of DNA content is shown. For comparison, top2-td curves from cells grown at the permissive temperature are indicated in pale gray for the diagrams of cells grown at the restrictive conditions.
Figure 3.
Cartoons illustrating the different patterns generated by the stereoisomers of undigested circular minichromosomes and linear DNA replication intermediates in 2D gels.
A: Linear forms (1.0x and 2.0x) and covalently closed monomers (CCCm) and dimers (CCCd) are depicted in black. CatA (catenanes where both rings are nicked) are depicted in light blue. CatB (catenanes where one ring is nicked and the other covalently closed) are depicted in red. CatC (catenanes where both rings are covalently closed) are depicted in green. KnCatA (nicked-catenanes where one or both rings are knotted) are depicted in yellow. And knotted monomers (Knm) are depicted in black encircled yellow. B: Complete patterns generated by linear replication intermediates. Bubbles in red, Simple-Ys in green, Double-Ys in violet, X-shaped recombinants in blue and unreplicated linear fragments in black. C: Patterns illustrating the transition from Bubbles to Double-Ys when it occurs after 1.5x. D: Patterns illustrating the transition from Bubbles to Double-Ys when it occurs before 1.5x. Note that here Double-Ys have a characteristic inflection. E and F: Patterns illustrating transitions from Bubbles to Simple-Ys.
Figure 4.
Analysis of replication products of pYAC_MEM and YAC_MEM with and without DNA topoisomerases.
Synchronized top2-td cells transformed with either pYAC_MEM or YAC_MEM were fixed 80 minutes after their release into the S-phase under permissive or restricted conditions. Undigested pYAC_MEM, the same DNA digested with the nicking enzyme NtBpU10I and undigested YAC_MEM DNAs were analyzed in 2D gels. The corresponding immunograms are shown together with a diagrammatic interpretation of the most prominent signals to their right. Cats = Catenanes A, B and C; OCms = Monomer Open Circles; Lms = Monomer Linears.
Figure 5.
Analysis of the replication intermediates of pYAC_MEM.
Synchronized top2-td cells transformed with pYAC_MEM were fixed 40 minutes after their release into the S-phase. The DNA was isolated, digested with the restriction enzymes shown to the left and analyzed in 2D gels. The corresponding immunograms are shown in the far left column with their corresponding interpretative diagrams to their right. Bubble arcs in red, simple-Y arcs in green and double-Ys in pale blue. Linear molecules and recombinants are shown in black. The simulation program 2D gel [19] was used to predict the shape of twelve consecutive RIs if replication proceeds unconstrained or if the leftward moving fork stalls permanently (Cen-P) or transiently (Cen-T) at the centromere CEN6. A linear map is shown on top of each series of RIs showing the relative positions of ARS4 (in green) and CEN6 (in magenta). The relative masses of the RIs are shown to the left. Red arrows indicate the transition mass of the RIs from bubbles to simple-Ys whereas blue arrows indicate transition mass from simple-Ys to double-Ys.
Figure 6.
Analysis of the replication intermediates of YAC_MEM in the presence and absence of DNA topoisomerases.
Synchronized top2-td cells transformed with YAC_MEM were fixed 40 minutes after their release into the S-phase under permissive or restricted conditions. The DNA was isolated and analyzed undigested in 2D gels. The corresponding immunograms are shown with their corresponding interpretative diagrams to their right. Bubble arcs in red and simple-Y arcs in green. Linear molecules and recombinants are shown in black. The simulation program 2D gel [19] was used to predict the shape of twelve consecutive RIs if replication initiated bi-directionally at ARS4 and proceeds unconstrained (shown to the left) or if initiation occurs at one telomere (shown to the right). A linear map is shown on top of each series of RIs showing the relative positions of ARS4 (in green) and CEN6 (in red). The relative masses of the RIs are shown to the left. The red arrow indicates the transition from bubbles to simple-Ys.
Figure 7.
Construction of pYAC_MEM_RFB+ and YAC_MEM_RFB+ and analysis of their replication intermediates.
A: Name, mass and genetic map of the circular minichromosome. The relative positions of its most relevant features are indicated inside: The centromeric sequence CEN6, the autonomous replication sequence (ARS4), URA3, the lambda DNA marker sequence (L1), the ribosomal RFB, Tetrahymena telomeric repeats, HIS3, the lambda DNA marker sequence (L2), the ColE1 unidirectional origin (ColE1 Ori) and the ampicillin-resistance gene (AmpR). Outside, the relative positions of sites recognized by specific restriction endonucleases are indicated. B: The corresponding linear map of the restriction fragment used and its size. Below, the genetic map of YAC_MEM_RFB+. C: Synchronized top2-td cells transformed with either pYAC_MEM_RFB+ or YAC_MEM_RFB+ were fixed 40 minutes after their release into the S-phase. The DNA was isolated, digested with the restriction enzymes shown to the left or kept undigested and analyzed in 2D gels. The corresponding immunograms are shown at the far left column with their corresponding interpretative diagrams to their right. Bubble arcs in red and simple-Y arcs in green. Linear molecules, recombinants and accumulated forms are shown in black. The simulation program 2D gel [19] was used to predict the shape of twelve consecutive RIs. For the BamHI-SwaI restriction fragment of the circular minichromosome pYAC_MEM_RFB+, if replication initiates at ARS4 and both forks proceed unconstrained (i), if replication initiates at ARS4, the leftward moving fork stalls permanently at CEN6 and the rightward moving fork moves unconstrained through the RFB (ii), and if replication initiates at ARS4, the leftward moving fork stalls transiently at CEN6 and the rightward moving fork stalls permanently either at the first or the second closely spaced sites of the RFB (iii). Below, for the linear minichromosome YAC_MEM_RFB+ on top, if replication initiates at ARS4 and both forks proceed unconstrained (i), if replication initiates at ARS4 and the rightward moving fork stalls permanently at the RFB (ii) and if replication initiates at the left telomere and the rightward moving fork stalls permanently at the RFB (iii). A linear map is shown on top of each series showing the relative positions of ARS4 (in green), CEN6 (in magenta) and the RFB (in red). The relative masses of the RIs are shown to the left. Red arrows indicate the transition mass of the RIs from bubbles to simple-Ys whereas blue arrows indicate the transition mass from simple-Ys to double-Ys.