Completion of replication map of Saccharomyces cerevisiae chromosome III

Abstract

In Saccharomyces cerevisiae chromosomal DNA replication initiates at intervals of approximately 40 kb and depends upon the activity of autonomously replicating sequence (ARS) elements. The identification of ARS elements and analysis of their function as chromosomal replication origins requires the use of functional assays because they are not sufficiently similar to identify by DNA sequence analysis. To complete the systematic identification of ARS elements on S. cerevisiae chromosome III, overlapping clones covering 140 kb of the right arm were tested for their ability to promote extrachromosomal maintenance of plasmids. Examination of chromosomal replication intermediates of each of the seven ARS elements identified revealed that their efficiencies of use as chromosomal replication origins varied widely, with four ARS elements active in < or = 10% of cells in the population and two ARS elements active in > or = 90% of the population. Together with our previous analysis of a 200-kb region of chromosome III, these data provide the first complete analysis of ARS elements and DNA replication origins on an entire eukaryotic chromosome.

Figure 1

Map of the rightmost 136 kb of chromosome III. Three overlapping segments of this region are shown with open reading frames indicated by arrows on the map lines and restriction sites used for subcloning indicated above the map lines. Open reading frames are based on the September 2000 release of the chromosome III sequence, and names indicated are from the Saccharomyces Genome Database (February 2001). Only restriction sites that mark the boundaries of subclones are shown; additional sites exist for each of these enzymes. The top segment extends from nucleotide 181,264-231,267, the middle segment from 226,264-276,191, and the bottom segment from 271,271 to the right end of the chromosome at nucleotide 316,613. The positions of ARS elements, identified by the subclones shown in this figure and further refined by subcloning shown in Figure 2, are indicated by shaded boxes. The open box at the right end of the lower segment indicates the position of the subtelomeric X, A, B, C, and D sequences (Louis et al., 1994). These subtelomeric sequences are followed by 93 bp of the G_1–3T telomeric repeat. Boxes below the map represent subclones prepared in the vector pRS306 and analyzed for ARS activity, with open boxes representing Ars⁻ subclones and filled boxes representing Ars⁺ subclones. With the exception of subclones YND58, YND60, RAD18, YND78, and YND79, described in Table 1, all subclones were derived from chromosome III inserts in bacteriophage λ or cosmid vectors prepared from strain AB972 by Riles and Olson (1993) and used in the chromosome III sequencing project (Oliver et al., 1992). Restriction enzyme abbreviations: A, AatII; B, BamHI; Bn, BstNI; Bs, Bst1107; Bw, BsiWI; C, ClaI; E, EcoRI; H, HindIII; K, KpnI; Pv, PvuII; Nc, NcoI; Rv, EcoRV; S, SalI; Sc, SacI; Sh, SphI; Sp, SpeI; Ss, SspI; X, XhoI; and Xb, XbaI.

Figure 2

Subcloning of ARS-containing regions. Features and restriction enzyme abbreviations are as indicated in the legend to Figure 1. (A) The 19-kb region extending from 180515–199513. Only a portion of the BPH1 open reading frame is shown. All subclones shown were derived from plasmid E5F (Newlon et al., 1991) except 188 and 205, which are subclones of plasmid 2-29 (Figure 1). (B) The 7-kb region extending from 220448 to 227511. Plasmids are subclones of 6-13 (Figure 1). YND96 contains a deletion of the Bst11071-SacI fragment shown. (C) The 9.7-kb region extending from 266905 to 276590. Only a portion of the YND63 subclone is shown. (D) The 7.7-kb EcoRI-BamHI fragment extending from 291306 to 299002 and containing HMR. a2 and a1 refer to the MATa2 and MATa1 transcripts encoded at HMR. (E) The 7.6-kb fragment containing the right end of chromosome III. Only a portion of the YND74 subclone is shown. The EcoRI site shown in parentheses is within a 14-bp segment of the m13 polylinker sequence that flanks the yeast DNA insert in the bacteriophage λ 2H4 clone from which the yeast fragment in plasmid YND78 was isolated (Louis, 1994).

Figure 3

2D gel analysis of chromosomal replication intermediates. ARS313: 3.6-kb EcoRI-HindIII fragment, probed with entire fragment; ARS314: 3.5-kb ClaI-EcoRV fragment, probed with 1.8-kb HindIII fragment. ARS315: 4.4-kb EcoRI fragment, probed with 1.3-kb SphI-BglII fragment; ARS316: 5.9-kb NheI-PstI fragment, probed with 2.5-kb BamHI fragment; ARS317 (HMR-E): 3.4-kb ClaI-BglII fragment, probed with 0.4-kb ClaI-EcoRI and 0.35-kb DraI-XbaI fragments; and ARS318 (HMR-I): 3.3-kb XbaI-EcoRV fragment, probed with 0.8-kb BglII fragment. Arrows point to faint bubble arcs. Black bars below the panels show the genomic fragments analyzed, and the boxes on the bars indicate the smallest subclone known to contain the ARS element. Hatched boxes indicate the probes used. DNA for the ARS317 and ARS318 patterns shown was from strain YPH45. DNA for the remainder of the patterns shown was from strain YPH47.

Figure 4

Direction of fork movement in chromosomal regions flanking ARS316, ARS317, ARS318, and ARS319. DNA was prepared from strain YPH45-7. Arrows above the arcs indicate the direction of fork movement. Maps below the top pairs of panels show the regions analyzed and gray boxes beneath the maps show the probes used. (A) Fragment left of ARS316: 5-kb XbaI-BamHI fragment cut in gel with PvuII and probed with a 2.3-kb PvuII-HindIII fragment. (B) Right of ARS316, a 6.3-kb HindIII fragment cut in gel with EcoRV and probed with a 2.1-kb EcoRV-SpeI fragment. (C) Left of HMR, a 4.8-kb HindIII fragment cut in gel with EcoRV and probed with a 2.3-kb HindIII-EcoRV fragment. (D) Right of HMR, a 5.7-kb SpeI fragment cut in gel with PvuII and probed with a 1.8-kb SpeI-SphI fragment. Arrows on the map show the a1 and a2 transcripts. (E) Left of ARS319, a 5.4-kb HindIII fragment cut in gel with EcoRV and probed with a 1.94-kb AatII-PvuII fragment from pRS306 as shown in F. (F) Schematic diagram of the right end of chromosome III in strainYP45-7. The plasmid YND95 (open box), a derivative of pRS306 which includes sequences from YCR105W and YCR106W, was integrated 3.8 kb from the beginning of the right telomere sequences. The hatched box represents pRS306 vector sequences, and ARS319 is shown as a black box. The probe used is indicated by the box below the map.

Figure 5

Summary of chromosome III replicator activity. ARS elements are numbered above the line. Rectangular boxes on the line show the positions of HML, MAT, and HMR. The filled circle shows the position of CEN3. The efficiencies of ARS elements as chromosomal replicators are indicated below the map line as the percentage of cell cycles in which they initiate replication. Values indicated as <10% identify ARS elements for which arcs of bubble-shaped replication intermediates have never been detected.