Modular nature of ARS elements. The functional elements defined by mutational analysis of four ARS elements are indicated by boxes on the line diagrams. All four are aligned by their essential matches to the ACS and drawn to the same scale. The tick marks below the lines indicate the positions +8, +10, +15, +17, and +18 relative to the end of the essential match to the ACS (see text). (A) ARS1 (12). (B) ARS307 (13, 14). (C) ARS305 (15). (D) ARS121 (16). The region labeled A+B1? corresponds to the “core” element of Walker et al. (16). ATR is the A+T-rich domain. B3-like corresponds to the pair of ARS binding factor 1 binding sites.

ORC footprinting of ARS309 and mutant derivatives. End-labeled fragments were footprinted with ORC. WT, wild-type ARS309 (Fig. 2A); 10/11, ARS309 with all four 10 of 11 matches mutated (Fig. 2F); 9/11, ARS309 with the essential 9 of 11 match mutated (Fig. 2G). For WT and 10/11, the four footprinting reactions contained 0, 5, 12.5, and 25 ng of ORC; for 9/11, the three reactions contained 0, 25, and 100 ng. Lanes R and T are chemical sequencing reactions for A+G, and T, respectively. The hatched boxes to the left indicate the protected regions (positions 60–102, lower box; 180–204, upper box), and the arrows point to hypersensitive sites (positions 87, 97, 196/197, 205/206 from bottom to top). Solid boxes to the right of the sequencing reactions mark the positions of the 10 of 11 matches to the ACS at positions 231–241 (upper box), 159–169 and 161–171 (middle box), and the essential 9 of 11 match at positions 62–72 (lower box). Arrows within these boxes indicate the orientation of the T-rich strand of the ACS. Note that neither the protected regions nor the hypersensitive sites are altered in the 10 of 11 match mutant, but the 9 of 11 match mutant abolishes the protection and hypersensitive sites in the region from positions 60 to 102.

Direction of replication fork movement in the left-flanking region of ARS309 and mutant derivatives. Genomic DNA was digested with PvuII and EcoRI before electrophoresis in the first dimension. Gel slices were excised, and the DNA was digested in situ with BglII prior to electrophoresis in the second dimension. (A) Wild-type ARS309. (B) Essential ACS mutant (Fig. 2G). (C) ARS309 with all 10 of 11 matches mutated (Fig. 2F). (D) ARS309 with nonessential 9 of 11 matches mutated (Fig. 2H). (E) Diagram of replication intermediates. When ARS309 is active, leftward-moving replication forks traverse the EcoRI–PvuII fragment (see F), and replication intermediates fall on the arc labeled L. When ARS309 is inactive, rightward-moving forks from ARS307 or ARS308 traverse the fragment, and the replication intermediates fall on the arc labeled R. (F) Line drawing indicating the position of relevant restriction sites and of the 0.35-kb HpaI–EcoRI fragment containing ARS309 (box). The 2.7-kb EcoRI–BglII fragment used as a probe is represented by the dashed line.

Effect of deletions and point mutations on ARS309 function. Lines represent the sequences present in plasmids. Position 1 is the first adenine of the HpaI site; position 349 is the cytidine of the EcoRI site. Solid boxes show the 10 of 11 matches to the ACS at positions 31–41, 159–169, 161–171, and 231–241. The open box marks the position of the 9 of 11 match at positions 62–72. The stippled boxes show the positions of the 9 of 11 matches at positions 75–85 and 94–104. × indicates point mutations altering the ACS matches (see Table 1). Boxes above the line indicate that the T-rich strand of the ACS is in the top strand, and boxes below the line indicate the T-rich strand of the ACS is in the lower strand. HFT: +, high-frequency transformation; −, no high-frequency transformation. Stability: Numbers show percentage of plasmid bearing cells in a logarithmic-phase culture under selection (mean ± SEM). (A) Full-length wild-type ARS309 (base pairs 1–349). (B) Deletion mutant containing base pairs 1–227. (C) Deletion mutant containing base pairs 1–147. (D) Deletion mutant containing base pairs 54–349. (E) RsaI subclone (base pairs 57–135). (F) Triple point mutation altering all four 10 of 11 matches (see Table 1). (G) Mutation altering the 9 of 11 match at positions 62–72 (see Table 1). (H) Mutation altering the two 9 of 11 matches at positions 75–85 and 94–104 (see Table 1).

Analysis of ARS309 replication intermediates. Genomic DNA was digested with BglII, electrophoresed, and blotted as described by Brewer and Fangman (5). The 3.6-kb BglII fragment containing ARS309 was detected by probing with the 1.4-kb HindIII–EcoRI fragment indicated by the dashed lines in C. The 0.35-kb HpaI–EcoRI fragment containing ARS309 is denoted by the box. (A) Wild-type ARS309. The arrows point to bubble-shaped replication intermediates, indicating that ARS309 is active as an origin on the chromosome. (B) ARS309 with the essential 9 of 11 match mutated (Fig. 2G). There are no bubble-shaped replication intermediates (arrows), demonstrating that origin function is abolished in the mutant.