Linear derivatives of Saccharomyces cerevisiae chromosome III can be maintained in the absence of autonomously replicating sequence elements

Abstract

Replication origins in Saccharomyces cerevisiae are spaced at intervals of approximately 40 kb. However, both measurements of replication fork rate and studies of hypomorphic alleles of genes encoding replication initiation proteins suggest the question of whether replication origins are more closely spaced than should be required. We approached this question by systematically deleting replicators from chromosome III. The first significant increase in loss rate detected for the 315-kb full-length chromosome occurred only after all five efficient chromosomal replicators in the left two-thirds of the chromosome (ARS305, ARS306, ARS307, ARS309, and ARS310) had been deleted. The removal of the inefficient replicator ARS308 from this originless region caused little or no additional increase in loss rate. Chromosome fragmentations that removed the normally inactive replicators on the left end of the chromosome or the replicators distal to ARS310 on the right arm showed that both groups of replicators contribute significantly to the maintenance of the originless chromosome. Surprisingly, a 142-kb derivative of chromosome III, lacking all sequences that function as autonomously replicating sequence elements in plasmids, replicated and segregated properly 97% of the time. Both the replication initiation protein ORC and telomeres or a linear topology were required for the maintenance of chromosome fragments lacking replicators.

FIG. 1.

Chromosome III replicators and loss rates of ARS deletion chromosomes. (A) Positions of ARS elements are indicated above the line representing chromosome III. ARS elements that function as efficient chromosomal replication origins are indicated as black boxes with white type. ARS elements that function in less than 25% of cell cycles are boxed with dotted lines (41). ARS elements that do not normally function as replication are not boxed.CEN3 is indicated by a filled circle on the map line. (B) ARS elements were deleted from the SUP11-1-marked chromosome of strain CF4-16B33 as described in Materials and Methods, and loss rates were determined by fluctuation analysis. The numbers within the bars indicate the last one or two digits of the names of the ARS elements deleted (e.g., 7 refers to ARS307). Error bars indicate standard deviations.

FIG. 2.

Loss rates of chromosomes fragmented near ARS304, ARS310, or both. Diagrams on the left show the 0ORIΔ derivatives of chromosome III used. 0ORIΔ, 5ORIΔ, and 6ORIΔ chromosomes described for Fig. 1 were fragmented as described in Materials and Methods. pYND104 was used for fragmentations near ARS304, producing the 283-kb fragment, and pYND56 was used for fragmentations near ARS310, producing the 174-kb fragment. The 141-kb derivative was fragmented at both ends. Loss rates were determined by fluctuation analysis. Error bars indicate standard deviations.

FIG. 3.

Two-dimensional gel analysis of replication intermediates of ARS301, ARS302, ARS303/ARS320, and ARS304 in 0ORIΔ and 6ORIΔ chromosomes fragmented near ARS310. DNA was prepared from strains CB08D3 (0ORIΔ) and YDN289 (6ORIΔ), which carry the S. carlsbergensis balancer chromosome. A diagram of the restriction fragment examined is shown below each pair of two-dimensional patterns. The filled rectangle represents the position of the ARS element(s). In all cases, the left panel of a pair shows the pattern obtained from the 0ORIΔ chromosome fragment and the right panel shows the pattern obtained from the 6ORIΔ fragment. A diagram of the 174-kb 6ORIΔ chromosome fragment examined is shown at the bottom of the figure. Landmarks on the map line are labeled as described for Fig. 2.

FIG. 4.

Analysis of the direction of replication fork movement along the 0ORIΔ and 6ORIΔ chromosomes fragmented near ARS310. DNA was prepared from strains described in the legend for Fig. 3. Diagrams of the restriction fragments analyzed to determine replication fork directions and their relationship to the ARS element or ARS deletion are shown below each set of three panels, with arrows showing the position(s) of the restriction sites used for in-gel digestion prior to the second dimension. The probes used are shown as thin lines below restriction fragment diagrams. In each set of three panels, the left panel shows the pattern obtained from the 0ORIΔ fragment, the middle panel shows the pattern obtained from the 6ORIΔ fragment, and the right panel is a diagram showing the arcs produced by leftward- and rightward-moving forks. How these replication intermediates are resolved depends on the geometry of the site of the in-gel digestion and the probe used to detect them. The directions of fork movement are indicated by arrows above the arcs in the diagrams. The diagram at the bottom of the figure summarizes the quantitation of directions of fork movement through the 6ORIΔ fragment. In this chromosome fragment, ARS308 was deleted by replacing CEN3 with CEN4, which is indicated by a filled circle on the map line. Hatched rectangles indicate the fragments analyzed, and the arrows beneath the hatched rectangles are marked with the percentage of forks going in each direction.

FIG. 5.

Diagrams of 61-kb chromosome III derivatives analyzed. (A) Ring chromosome III (61 kb) formed by recombination between Ty1-17, which is a Ty2 element, and Ty1-161 (Fig. 1A). The hybrid Ty formed by the recombination is designated “Ty” on the maps in the figure. (B) Ring chromosome (61 kb) carrying the 276-bp TRS as described in Materials and Methods. (C) The ring chromosome was linearized as described in Materials and Methods.

FIG. 6.

Stabilities of 0ORIΔ and 5ORIΔ chromosomes fragmented near ARS310 or near ARS304 and ARS310 in orc5-1 and orc2-1 strains. Diagrams of the 174-kb and 142-kb 0ORIΔ fragments are shown at the top of the figure. Strains YKN6 (wild type), YNS1 (orc2-1), and YNS3 (orc5-1) were mated with donor strains F013 and F510, and chromoductants carrying the chromosome III 0ORIΔ and 5ORIΔ fragments were selected as described in Materials and Methods. Loss rates were determined by fluctuation analysis. Loss rates in the wild-type and orc2-1 strains were determined at 23°C, and loss rates in the orc5-1 strain were determined at the semipermissive temperature of 27°C. Error bars indicate standard deviations.