Analysis of chromosome III replicators reveals an unusual structure for the ARS318 silencer origin and a conserved WTW sequence within the origin recognition complex binding site

Abstract

Saccharomyces cerevisiae chromosome III encodes 11 autonomously replicating sequence (ARS) elements that function as chromosomal replicators. The essential 11-bp ARS consensus sequence (ACS) that binds the origin recognition complex (ORC) has been experimentally defined for most of these replicators but not for ARS318 (HMR-I), which is one of the HMR silencers. In this study, we performed a comprehensive linker scan analysis of ARS318. Unexpectedly, this replicator depends on a 9/11-bp match to the ACS that positions the ORC binding site only 6 bp away from an Abf1p binding site. Although a largely inactive replicator on the chromosome, ARS318 becomes active if the nearby HMR-E silencer is deleted. We also performed a multiple sequence alignment of confirmed replicators on chromosomes III, VI, and VII. This analysis revealed a highly conserved WTW motif 17 to 19 bp from the ACS that is functionally important and is apparent in the 228 phylogenetically conserved ARS elements among the six sensu stricto Saccharomyces species.

FIG. 1.

Structure of yeast replicators. ARS1 (26), ARS307 (37, 46), and ARS315 (12) are diagrammed, highlighting the A, B, and C regions important for replicator activity. Region A contains the ACS, and region B is further defined by modular B1, B2, and B3 (Abf1) elements. ACS and B1 together comprise the ORC binding site. I^S depicts an inhibitory element within a positioned nucleosome. Some replicators contain transcription factor binding sites in region C that stimulate their activity (not shown).

FIG. 2.

Identifying the ACS for ARS318. Various deletions and point mutations within ARS318 sequences were constructed and tested for their ability to confer ARS activity by transformation into W303-1A. HFT indicates high frequency transformation. The loss rates of these same plasmids were quantitated using a plasmid loss assay (right). The sequence at bottom indicates the positions of the SalI linker mutations as well as deletions Δ3 and Δ4 within ARS318. The shaded sequence represents the DNase I Abf1p footprint from reference , and the arrows indicate the 9/11- and 10/11-bp matches to the ACS.

FIG. 3.

Structure of ARS318. The structure of ARS318 was determined by assaying 29 derivatives of pFJ13 carrying 7-bp SalI linker scan mutations for their ability to transform W303-1A and also for their plasmid loss rate. Wild-type (WT) ARS318 was lost at a rate of about 4.5% per generation. The boxed regions within the DNA sequence indicate the functional regions delimited by the linker scan mutations and correspond to the Abf1p binding site, the ACS (A), and the B1 and B2 elements. Plasmids containing mutations in the ACS could not be assayed, since they fail to yield transformants. Plasmid loss rates represent the averages ± SEM of results from at least six independent measurements.

FIG. 4.

ARS318 is an active replicator in the absence of ARS317. (A) The right end of chromosome III is diagrammed, highlighting the ARS317, ARS318, and ARS319 replicators. The arrow at the right indicates the direction of forks that replicate this region emanating from ARS319, and the shaded box indicates the extent of the 800-bp HMR-E deletion. (B) Southern blots of 2D gels showing ARS318 replicator activity in the wild-type (W303-1B) and ΔHMR-E (CFY2133) strains.

FIG. 5.

Identifying the ACS for ARS313, ARS316, ARS317, ARS318, and ARS319. (A) ARS elements were cloned into a URA3 CEN4 plasmid (Table 1) and then tested for wild-type (WT) ARS activity by transformation of W303-1A and selection for Ura⁺ transformants. The same plasmids containing the indicated SalI linker mutations within the essential ACS (described in the text) eliminated ARS activity by the same ACS⁻ transformation assay. (B) ARS317 (HMR-E) contains a redundant ACS 3′ to the silencer. pFJ11 (URA3 CEN4) containing the HMR-E silencer or various mutants (diagrammed at bottom) were transformed into W303-1A and assayed for growth at 30°C. The 11/11-bp match to the ACS (site E) is defined as the ACS element within the silencer. The five 9/11-bp matches to the ACS within the B-region of the ARS317 replicator are indicated as arrows, and mutations A to D disrupt these sequences (see the text for additional details).

FIG. 6.

(A) Alignment of 32 ARS elements on chromosomes III, VI, and VII by use of WebLogo reveals the EACS and a conserved WTW motif within B1. The ACS is numbered from +1 to +11. See the text for details. (B) WTW is conserved among the 228 phylogenetically conserved ARS elements. The 228 ARS elements conserved in the six sensu stricto Saccharomyces species (32) were aligned using WebLogo (13) and shown to conserve a WTW motif within the B1 element.

FIG. 7.

The WTW sequence is important for origin activity at multiple ARS elements. (A) The DNA sequence surrounding the ACS and B1 elements are shown for six ARS elements. The essential ACS is underlined, and the WTW sequence within B1 is shaded. (B) The wild-type ARS plasmids (light gray bars) and the corresponding WTW→WGG derivatives (dark gray bars) were transformed into W303-1A and quantitated for their plasmid loss rates. Two wild-type ARS316 plasmids (pRF32 and pFJ23) were assayed together with their respective WGG derivatives. Plasmid loss rates represent the averages ± SEM of results from at least six independent measurements.

FIG. 8.

WTW is the critical sequence within the ARS318 B1 element. A series of 2-bp linker scan mutations spanning the B1 region of ARS318 defined by the 7-bp SalI linkers in Fig. 3 were constructed. Mutation of the WTW base pairs but not adjacent nucleotides substantially decreased ARS activity as determined by a plasmid stability assay, as in Fig. 7. WT, wild type.