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

Saccharomyces cerevisiae chromosome III encodes 11 autonomouslyreplicating sequence (ARS) elements that function as chromosomalreplicators. The essential 11-bp ARS consensus sequence (ACS)that binds the origin recognition complex (ORC) has been experimentallydefined for most of these replicators but not for ARS318 (HMR-I),which is one of the HMR silencers. In this study, we performeda comprehensive linker scan analysis of ARS318. Unexpectedly,this replicator depends on a 9/11-bp match to the ACS that positionsthe ORC binding site only 6 bp away from an Abf1p binding site.Although a largely inactive replicator on the chromosome, ARS318becomes active if the nearby HMR-E silencer is deleted. We alsoperformed a multiple sequence alignment of confirmed replicatorson chromosomes III, VI, and VII. This analysis revealed a highlyconserved WTW motif 17 to 19 bp from the ACS that is functionallyimportant and is apparent in the 228 phylogenetically conservedARS elements among the six sensu stricto Saccharomyces species.

INTRODUCTION

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INTRODUCTION

Chromosomal origins of DNA replication in budding yeast arecalled autonomously replicating sequence (ARS) elements andwere identified about 30 years ago by their ability to conferautonomous replication to originless plasmids (30). A conserved11-bp sequence called the ARS consensus sequence (ACS) was initiallyidentified by the analysis of the DNA sequences of four ARSelements (8). The ACS is now known to comprise a binding sitefor the origin recognition complex (ORC), the essential initiatorprotein in all eukaryotes (reviewed in reference 2). The consensussequence of the AT-rich ACS element (WTTTAYRTTTW) is degenerate,and bona fide ARSs sometimes contain only a 10/11- or 9/11-bpmatch to this sequence. A W indicates the base A or T. Whenadditional ACSs were identified experimentally, a 17-bp extendedACS (EACS) was defined and reflected that the bases flankingthe ACS were often A's or T's (WWW-ACS-[G/T]WW) (47). Althoughthe ACS is essential for replicator activity, it cannot be thesole determinant of ORC binding and/or origin specification.By pattern matching, there are 860 exact matches to the 11-bpACS and 13,978 ACSs, allowing one mismatch in the yeast nucleargenome (http://seq.yeastgenome.org/cgi-bin/PATMATCH/nph-patmatch).Since there are only $~$ 350 functional ARS elements in Saccharomycescerevisiae, excluding the ribosomal DNA locus (25), additionalsequences, chromatin environment, active transcription units,higher-order nucleosome structure, and/or nuclear organizationmight further restrict the locations of functional ARS elements.In agreement with one of these predictions, genome-wide studiesof ORC and MCM binding sites revealed that >90% of putativeARSs are intergenic (50, 51). Furthermore, additional sequencesin the B region adjacent to the ACS (Fig. 1) are known to contributeto ORC binding and origin activity in S. cerevisiae (26, 37,41, 46).

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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).

Detailed analysis of ARS1 (26) and ARS307 (37, 46) showed thatthey have modular structures (Fig. 1). In addition to the ACS(A element), they also contain B1 and B2 elements 3' to theT-rich strand of the ACS. The ARS1 B region additionally containsan Abf1p binding site, also called the B3 element. ARS302 (43),ARS305 (12, 22), and ARS315 (12) contain B1 and B2 elementsas well, and studies of several chromosome VI ARS elements alsodefined domain B elements that were important for origin activity(39). The B1 sequences at ARS1 and ARS307 include sequencesimportant for ORC binding (38, 41), and it is now recognizedthat ORC contacts a bipartite DNA sequence consisting of nucleotidesin the ACS and B1 elements (21). Some ARS elements also containstimulatory sequences 5' to the T-rich strand of the ACS indomain C. For several of these ARS elements, domain C containstranscription factor binding sites for Abf1p (49), Rap1p (43),Mcm1p (11), or Sum1p (20) that stimulate, but are not requiredfor, origin activity. Recently, an ARS inhibitory element thatlikely influences local chromatin structure was discovered 3'to the B2 element in ARS305 and ARS315 (12).

Because the sequence characteristics of the B1 and B2 elementshave not been experimentally defined at multiple replicatorsand because there may be as-yet-undefined regulatory elements,we are analyzing additional replicators on chromosome III toidentify their functional sequences. Chromosomes III and VIwere the first entire chromosomes to be analyzed systematicallyfor the presence of ARS elements. ARS elements were identifiedby their ability to confer autonomous plasmid replication andthen confirmed as chromosomal replicators by neutral-neutraltwo-dimensional (2D) gel analysis to detect origin activityat their endogenous chromosomal locations (18, 29, 36, 44, 52).Chromosome III contains 11 active replicators, and chromosomeVI contains 10.

Here, we have completed the identification of the essentialACS(s) of the replicators on chromosome III by analyzing ARS313,ARS316, ARS317, ARS318, and ARS319 and have determined the detailedstructure of the silent mating-type replicator ARS318 (HMR-I).ARS318 has an A-B1-B2 structure but also contains an Abf1p bindingsite 6 bp 5' to the ACS that contributes to its activity. AlthoughARS318 is largely inactive on the chromosome, we found thatARS318 becomes active when the adjacent HMR-E silencer is deleted.Furthermore, although the 11/11-bp match to the ACS of ARS317is a well-studied ORC binding site, we identified a redundantACS that functions in its absence. ARS313, ARS316, and ARS319each contained a single essential ACS. Analysis of multipleactive origins on chromosomes III, VI, and VII revealed a conservedand highly significant WTW motif present within the B1 elementthat is very important for replicator function. This sequenceis also conserved (P $≤$ 1 x 10^–10) within the 228 ARS elementsphylogenetically conserved among the six sensu stricto Saccharomycesspecies (32). Previous studies have noted a bias toward AT basepairs in this region (6, 30, 51). Therefore, when comparativeanalysis was confined to active or phylogenetically conservedreplicators, the conserved WTW motif was revealed to be highlysignificant. This conserved sequence within B1 likely makesimportant ORC-DNA contacts at many replicators based on previousORC-ARS1 and ORC-ARS307 binding studies (21, 38, 41) and ourphenotypic analysis of WTW mutants. The ACSs for all activereplicators on chromosome III have now been defined experimentally,with the exception of ARS308, which overlaps the CEN3 centromere.In addition, the detailed structures of ARS1 (26), ARS305 (12,22), ARS307 (37, 46), ARS315 (12), and ARS318 are now known,allowing for some generalization about replicator structure.

MATERIALS AND METHODS

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MATERIALS AND METHODS

Yeast strains and methods. The yeast strains used in this study were W303-1A (MATa ade2-1trp1-1 ura3-1 leu2-3,112 his3-11,15 can1-100), W303-1B (MAT ${alpha}$ ade2-1 trp1-1 ura3-1 leu2-3,112 his3-11,15 can1-100) (48), andCFY2133, which is W303-1B containing an 800-bp deletion of theHMR-E locus (10, 28). Yeast transformation and culturing wereperformed according to standard methods. YPD medium contains20 g yeast extract, 10 g peptone, and 20 g dextrose per liter.Synthetic complete medium (SCM) contains 50 g dextrose and 6.7g yeast nitrogen base without amino acids (Difco) per literand is supplemented with 20 µg/ml uracil and adenine andthe 20 amino acids.

Plasmid construction. All wild-type chromosome III ARS elements were cloned into thepARS1-WT (CEN4 URA3) backbone (26), replacing ARS1, and weredescribed previously (12), with the exception of pFJ23 and pRF23.A 318-bp ARS316 fragment (chromosomal coordinates 272768 to273085) was amplified with EcoRI-HindIII sites and cloned intothe same sites of pARS1-WT to give pFJ23. pRF23 similarly hasARS318 chromosomal coordinates 294567 to 295148 cloned on anEcoRI-HindIII fragment into pARS1-WT. The deletion derivativeswithin ARS318 (Fig. 2) were made by QuikChange mutagenesis asfollows: pFJ10 ( ${Delta}$ 1) deletes 94 bp from the EcoRI site of pRF23,pVMH40 ( ${Delta}$ 2) deletes 194 bp from the EcoRI site of pRF23, pVMH42( ${Delta}$ 3) deletes 252 bp from the EcoRI site of pRF23, pFJ12 ( ${Delta}$ 4) deletes294 bp from the EcoRI site of pRF23, pFJ14 ( ${Delta}$ 5) deletes 143 bpfrom the HindIII site of pVMH40, and pFJ13 ( ${Delta}$ 6) deletes 185 bpfrom the HindIII site of pVMH40. pFJ157 contains chromosomalcoordinates 292134 to 292652 directly to the left (centromereproximal region) of HMR-E on an EcoRI-HindIII fragment in pARS1-WT.pFJ166 is pFJ157 containing an XhoI linker (CCTCGAG) that eliminatesthe two overlapping 9/11-bp matches to the ACS in site A ofFig. 5B. All other plasmid derivatives used in the study deviatefrom the wild-type sequence as shown in the figures.

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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 ${Delta}$ 3 and ${Delta}$ 4 within ARS318. The shaded sequence represents the DNase I Abf1p footprint from reference 9, and the arrows indicate the 9/11- and 10/11-bp matches to the ACS.

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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).

Plasmid stability assay. We performed the plasmid stability assays essentially as describedpreviously (35). At least six independent cultures were inoculatedfrom single colonies and grown overnight at 25°C in SCMwithout uracil for $~$ 18 h. The cultures were diluted 1:2,000 intoSCM and grown for 24 h at 25°C, during which time they underwentapproximately 10 doublings in the absence of selection for theARS plasmid. Dilutions of both overnight cultures were platedonto SCM and SCM-Ura plates, and colonies were counted 72 hlater. Generational loss rates were calculated, and standarderrors of the mean (SEM) are reported for each assay.

Alignment of chromosome sequences and statistical analysis. Active origins on chromosomes III, VI, and VII were compiledwith the ACS elements aligned, and the sequence logos were generatedusing a web-based application, WebLogo (13) (http://weblogo.berkeley.edu/).The WebLogo sequences of 228 confirmed ARS elements identifiedby Nieduszynski et al. (32) were also generated using the alignedACS elements. The statistical significance of the proportionof WTW hits in the 228 ARS elements at positions 19 to 21 distalto the ACS was estimated by first creating 1 million samples,each containing 228 random 31-mer sequences selected from thewhole genome. For each sample, the proportion of the 228 sequencescontaining WTW in the equivalent position was calculated. Themean and standard deviation for these million samples approximatesthe mean and standard error for the expected proportion. TheP value was estimated based on a normal approximation for theproportions, which was reasonable based on a visual inspectionof the distribution for the random samples.

Determining conservation of WTW motif across the sensu stricto species. Forty-two phylogenetically conserved and confirmed S. cerevisiaereplicators that had at least 12 out of 15 base identities withinthe proposed 15-bp ACS motif (proACS) (32) as determined bycomparing Saccharomyces paradoxus, S. mikatae, S. kudriavzevii,and S. bayanus to S. cerevisiae were selected (see Fig. S2 inthe supplemental material). The alignments among the sensu strictospecies for each of these ACS elements can be seen at OriDB(Origin Database) (31). For each replicator and for each species,the proACS motif was retrieved with the addition of 30 bp 5'and 30 bp 3'. For each species, the resulting 42 sequences wereused to generate the WebLogo diagrams in Fig. S3 in the supplementalmaterial.

Analysis of replication intermediates. DNA was extracted from log-phase cultures of W303-1B and CFY2133as described previously (7). Sixty micrograms of DNA was thendigested with XbaI-EcoRV and subjected to BND-cellulose (Sigma)chromatography as described in reference 15, except that volumeswere cut in half. Neutral-neutral 2D agarose gels were blottedto Nytran SPC (Whatman) and hybridized as described previously(46). Membranes were probed with the 1-kb XbaI-BglII fragmentof pYND70 (36), which was labeled with [ ${alpha}$ -³²P]dATP (Perkin Elmer),using the Megaprime DNA-labeling system (GE Healthcare).

RESULTS

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RESULTS

Identifying the ACS for ARS318. We cloned 14 ARS elements from chromosome III into the sameCEN4 URA3 reporter plasmid (12) and determined their loss ratesin W303-1A (Table 1). Most loss rates varied from $~$ 3 to 8% pergeneration except that ARS302, ARS306, ARS314, and ARS316 werelost at higher rates, from 12 to 30% per generation. ARS318,which coincides with the HMR-I silencer near the right end ofchromosome III, is largely inactive as a chromosomal replicator(40). However, a plasmid carrying a 583-bp insert containingARS318 was lost at $~$ 4.5% per generation (Fig. 2), indicatingthat ARS318 is an efficient replicator when removed from itsnative chromosomal context (1, 8). Highly efficient chromosomalreplicators, such as ARS1, which are active in nearly everycell cycle, have similar low plasmid loss rates. Successive $~$ 50-bp deletions from the left (telomere distal) end of the 583-bpARS318 fragment did not alter plasmid stability (Fig. 2, ${Delta}$ 1 and ${Delta}$ 2). However, further deletion of the Abf1p binding site (9)caused a fourfold increase in the plasmid loss rate (Fig. 2, ${Delta}$ 3). Removal of an additional 41 bp eliminated replicator activity(Fig. 2, ${Delta}$ 4), indicating that sequences within this 41-bp regionwere essential for ARS318 function. We then deleted telomere-proximalsequences and defined a $~$ 200-bp fragment that was maintainedas well as the original 583-bp ARS318 fragment (Fig. 2, ${Delta}$ 6).

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TABLE 1. Loss rates for chromosome III ARS plasmids

There are three 9/11-bp matches and one 10/11-bp match to theACS within the smaller ARS318 plasmid pFJ13. To determine sequencesimportant for ARS activity, we made 7-bp SalI linker substitutionmutations (GGTCGAC) within each potential ACS and in the Abf1pbinding site (Fig. 2). Mutation of the Abf1p site increasedthe plasmid loss rate approximately threefold, indicating thatthis site, which is known to bind Abf1p, contributes significantlyto ARS activity. The 9/11-bp match directly adjacent to theAbf1p site was essential for ARS function, as shown by the failureof plasmids carrying this mutation to produce Ura⁺ transformants.In contrast, mutation of the three remaining putative ACSs causedat most a twofold increase in the plasmid loss rate. Thus, the10/11-bp match to the ACS is not essential for ARS activity,as was suggested based on its higher degree of similarity tothe ACS (8). However, sequences within the 10/11-bp match tothe ACS likely contribute to ARS function by encoding the B2element (next section).

ARS318 has a modular Abf1-A-B1-B2 structure. Since the organization of the ARS318 replicator appeared tobe different from that of known ARS elements, we performed alinker scan analysis of ARS318 in pFJ13 to determine the positionsof its functional elements. We created a series of 29 plasmidsthat contained ordered 7-bp SalI linker mutations and testedtheir ability to transform W303-1A to Ura⁺ (Fig. 3). All ofthe plasmids transformed the wild-type strain with high frequenciesexcept for pFJ32 and pFJ42, which carry mutations in the 9/11-bpmatch to the ACS described above. We conclude that the TATCATGTTTTsequence corresponds to the functional ACS at ARS318. Anothersequence located between 16 and 26 bp 3' to the ACS was alsoimportant for ARS function (Fig. 3). However, mutation of sequencesfrom base pairs +16 to +22 had a much greater effect on ARSactivity (41% loss rate) than mutation of base pairs +23 to+26, which increased the loss rate about twofold, to 10%. Thepositions of the sequences 16 to 26 bp from the ACS correspondto the B1 elements of ARS1, ARS305, ARS307, and ARS315. Twoadditional linker scan mutations 23 bp and 30 bp distal to B1caused a twofold decrease in plasmid stability. These overlapthe 10/11-bp match to the ACS and correspond to the B2 elementsdefined in the four replicators cited above. Finally, mutationsin the Abf1p binding site increased the plasmid loss rate two-to threefold (Fig. 3). There were no other sequences surroundingARS318 that promoted or inhibited ARS activity.

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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.

ARS318 is an active chromosomal replicator in the absence of the HMR-E silencer. Since ARS318 was an efficient replicator in a plasmid contextbut largely inactive on the chromosome, we thought its chromosomalposition might render it inactive. ARS317 and ARS318 flank thetranscriptionally silent a-type mating genes on the right endof chromosome III about 22 kb from the telomere. ARS318 is proximalto the telomere, and the entire HMR region is replicated in $~$ 90% of cell cycles from leftward-moving forks that originatefrom the subtelomeric replicator ARS319 (36) (Fig. 4A). ARS317replicates this region in the remaining 10% of cell cycles.Therefore, proximity to the telomere, the nearby highly activeARS319 replicator, the local heterochromatin at the HMR silencer,or the adjacent ARS317 replicator could potentially inhibitARS318 activity. We tested the latter two possibilities by deletingHMR-E on the chromosome and examining ARS318 activity by 2Dgel electrophoresis. Under this condition, both the adjacentARS317 replicator and the heterochromatic environment are eliminated,since HMR-E is essential for heterochromatin formation at theHMR locus (42). In the wild type, HMR-I was largely inactive,as indicated by the strong Y arc (passive replication from ARS319)and the very faint bubble arc (Fig. 4B), as previously shown(40). However, when HMR-E was deleted, ARS318 then became anactive replicator, as evidenced by the strong bubble arc. Weestimate that ARS318 is active in more than 50% of cell cycles.These data suggest that ARS317 or the heterochromatic environmentat HMR (or both) inhibits ARS318 activity.

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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 ${Delta}$ HMR-E (CFY2133) strains.

Determining the ACSs for ARS313, ARS316, and ARS319. We cloned ARS313 and ARS319 into the CEN4 URA3 plasmid on 287-bpand 393-bp fragments, respectively. ARS313 and ARS319 fragmentscontain single 10/11-bp and 11/11-bp matches to the ACS, respectively,and both plasmids were maintained very efficiently (Table 1).A 7-bp SalI substitution mutation within each putative ACS resultedin a loss of ARS activity (Fig. 5A). Thus, these sequences areessential for ARS activity and likely represent the ACS forthese replicators. The situation for ARS316 was more complexbecause its sequence had not been narrowly defined; ARS316 hadbeen mapped to an $~$ 1,185-bp fragment (36). We cloned ARS316 ona 502-bp fragment that contained five 9/11-bp matches to theACS (but no 10/11- or 11/11-bp matches) (see Fig. S1 in thesupplemental material). The pFJ23 plasmid, carrying a deletionof 184 bp from one end of this clone, removed three potentialACSs but retained ARS activity, indicating that pFJ23 containedthe essential ACS. Sequences within this $~$ 184-bp fragment stimulatedARS activity since the loss rate increased from 13% to 16.8%.Mutation of one 9/11-bp-match ACS (TTTCAAATTTA) abolished originactivity within pFJ23 (Fig. 5A) and in the context of the larger,502-bp fragment in pRF32 (see Fig. S1 in the supplemental material).Mutation of the other 9/11-bp-match ACS allowed high efficiencytransformation of yeast and, therefore, was not essential forARS activity (see Fig. S1 in the supplemental material). Thus,this single 9/11-bp match to the ACS is required for ARS316replicator activity. Our data for ARS313, ARS316, and ARS319are in agreement with the results of a recent genome-wide studyof phylogenetically conserved ARS (EACS) elements (32).

Redundancy in the ARS317 replicator. The HMR-E silencer on the right end of chromosome III has replicatoractivity corresponding to ARS317. ARS317 is active in $≤$ 10% ofcell cycles in its chromosomal context (40) but is an efficientreplicator in a plasmid context (Table 1) (1). Therefore, itschromosome position or additional chromosomal sequences contributeto its relative inactivity within its native context. An 11/11-bpmatch to the ACS in ARS317 binds ORC (3, 34). However, mutationof the ACS abolishes ARS activity only within a minimal 138-bpfragment containing the silencer elements, i.e., the ORC, Rap1p,and Abf1p binding sites (Fig. 5B) (19, 28, 33). In other words,if natural sequences flanking this 138-bp minimal fragment arepresent, the ACS^– mutant retains replicator activity bothin the plasmid and on the chromosome. The residual origin activityis due to activation of redundant nearby origins, which arenormally suppressed by the HMR-E silencer (19, 33).

We cloned a 345-bp fragment containing HMR-E and determinedthe sequences required for ARS activity, using SalI linker mutagenesisas described above. Confirming previous studies, mutation ofthe 11/11-bp match to the ACS reduced but did not eliminateARS activity, as evidenced by the appearance of slower-growingUra⁺ transformants (Fig. 5B, mutant E). There are five 9/11-bpmatches to the ACS within 80 bp of the ORC binding site, correspondingto the B region of ARS317. We made SalI mutations that eliminatedthe 9/11-bp matches; however, none of these mutations (A toD) affected the transformation frequencies of the plasmids orthe sizes of the resulting Ura⁺ colonies (data not shown). Thisindicated that sites A to E were not essential for ARS activity.In contrast, mutations in sites A or B when combined with thesite E mutation caused the formation of tiny Ura⁺ transformantsthat could not be propagated (Fig. 5B). Two overlapping 9/11-bpmatches to the ACS were affected by the A and B mutations; mutationA affects both potential ACSs but mutation B only the second.Therefore, the second 9/11-bp match to the ACS probably confersARS activity in the absence of the primary ORC binding site.

A weak ARS activity that functions only in the absence of ARS317has been mapped within $~$ 500 bp to the left of HMR-E (40). Ouranalogous plasmid clone pFJ157 also had weak ARS activity, whichwas eliminated by mutation A, affecting the overlapping 9/11-bp-matchACSs (Fig. 5B, pFJ166). Therefore, these 9/11-bp ACS matchesare required for this ARS activity. Since plasmid p ${Delta}$ 173 begins40 base pairs to the left of the 9/11-bp matches and lacks ARSactivity (1) (Fig. 5B), this also confirms that the essentialsequences for the left ARS activity map to this region.

Alignment of known chromosomal ARS elements reveals a WTW motif within B1. Since we determined the ACSs for four more ARS elements on chromosomeIII in this study and also recently analyzed the structuresof ARS305 and ARS315 (12), we aligned the active chromosomeIII, VI, and VII ACSs and looked for additional conserved sequencesflanking the ACS by using WebLogo (13) (Fig. 6A). We numberedthe 11-bp ACS from +1 to +11. As expected, we found a selectionfor WWW at residues –1, –2, and –3 from theACS and (G/T)TT at positions +12, +13, and +14, which matchedthe EACS (47). In addition, we saw a strong selection for WTWbeginning 17 bp 3' to the ACS (positions +28 to +30). This sequencefalls within the B1 elements of ARS1, ARS302, ARS305, ARS307,ARS315, and ARS318. Interestingly, these nucleotides were conservedat levels similar to key nucleotides within the EACS. A biastoward AT base pairs in this region was previously noted basedon alignment of known origins (47) and also computational predictionand evaluation of ARS elements (6). However, the enrichmentthat we observed in these 32 active chromosomal origins wasmore highly significant.

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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.

This result prompted us to look for the WTW sequence in the228 evolutionarily conserved ARS elements within the sensu strictoSaccharomyces species (32). These six species diverged withinthe last 35 million years but retain the ability to mate witheach other, yielding sterile diploids (24). The conservationof these ARS elements is a strong indicator that they are functionalreplicators across species, and this supposition was directlydemonstrated using a plasmid ARS assay with S. cerevisiae forthe 228 replicators (32). Alignment of the 228 S. cerevisiaeARS elements showed once again a highly significant enrichmentof WTW 17 to 19 bp distal to the ACS (Fig. 6B), with no furtherconserved sequences at least 30 bp 3' to the WTW sequence (datanot shown). The P value for the chance occurrence of the WTWsequence was $≤$ 1 x 10^–10 in comparison to a similar enrichmentof this sequence within the genome. Interestingly, the WTW motifwas also apparent among the 42 most highly conserved ACS elementsacross the sensu stricto species (see Fig. S2 and S3 in thesupplemental material).

WTW is important for ARS activity but is not required at the silencer origin ARS317. The conservation of the WTW sequence near the ACS, and the factthat nucleotides within this region affect ORC binding at ARS1and ARS307, strongly suggested that these nucleotides wouldbind ORC at other replicators and influence ARS activity invivo. We tested this directly at six chromosome III ARSs bymutating WTW to WGG (Fig. 7A) and then measuring plasmid lossrates for the resulting mutants compared to the wild-type levels.For ARS313, ARS315, ARS316, and ARS318, mutation of this sequencedramatically increased the loss rate to 30% per generation orhigher (Fig. 7B). We could not accurately measure the loss ratesfor ARS316 mutants, because they grew very poorly in liquidmedium, but the loss rates had to be greater than 35% sincethat rate is measurable. The WGG mutation at the subtelomericARS319 mutant increased plasmid loss rate twofold (Fig. 7B).In contrast, mutation of WTW at ARS317 did not affect its stability.Interestingly, ARS317 binds ORC 10-fold more tightly than efficientorigins such as ARS1 (34). Our data therefore suggest the possibilitythat mutation of WTW at ARS317 does not sufficiently disruptORC binding (or subsequent ORC-dependent steps in origin activation)to alter origin activity. At the other origins tested, mutationof this site strongly destabilized origin function, indicatingthat the conservation of this sequence is functionally relevant.

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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.

We performed a 2-bp linker scan over the ARS318 B1 region todetermine if there were sequences other than WTW that were importantfor replicator activity (Fig. 8). Mutation of the WTW nucleotidesdecreased ARS activity from three- to eightfold; however, mutationof nucleotides directly adjacent to WTW had little effect. Mutationof nucleotides 5 to 8 bp distal to WTW decreased ARS activitymodestly ( $~$ 70 to 80%), but no other nucleotides were importantfor ARS activity. Thus, at ARS318 the WTW sequence is the mostimportant determinant of the B1 element.

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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.

ACS plus WTW is a better predictor of ARS element location than the ACS alone. We found that the ACS plus the WTW sequence was a better searchstring for identifying known ARSs. We searched chromosome Xfor matches to the ACS (including 1 bp on either side) withor without the WTW sequence in B1 and identified 3,577 matchesto the 13-bp ACS (up to 2-bp mismatches) and 994 matches byincluding the WTW sequence. Both sets included 15 of the 16known chromosome X ARS elements with conserved ACSs (32), indicatingthat the search strings had good sensitivity but ACS plus WTWimproved the stringency since it eliminated about three-fourthsof the false hits. However, even the ACS-plus-WTW search stringwas not stringent enough, because it identified 979 additionalmatches, most of which were probably not ARSs. Limiting thesearch to intergenic sequences did not improve the stringency(data not shown); however, it reduced the number of hits, asexpected. Lastly, searching with a 15-bp ACS (including 2 bpon either side of the ACS) with or without WTW did not significantlyalter the above-described outcome. Therefore, the ACS plus theWTW sequence is a better predictor of known replicators butadditional genetic or epigenetic information must determinethe positions of ARS elements in budding yeast.

DISCUSSION

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DISCUSSION

The ACS is conserved at yeast replicators because it is a bindingsite for the initiator complex ORC (2). Point mutations withinthe ACS or B1 element that disrupt ORC binding also impair ARSactivity. In addition, the spacing of the B1 element and theACS is critical for ORC binding. Deletion of one or more basepairs between the ARS603 ACS and B1 elements causes impairedORC binding in vitro (4). Therefore, it is not surprising thatmultiple replicators have defined B1 elements similarly positionedwith respect to the ACS. The B1 element is located within 10to 31 bp 3' to the ACS, as defined by 7- or 8-bp linker scanmutations (12, 26, 37, 46). By use of 3-bp linkers at ARS307,the B1 sequence has been more narrowly defined and containscritical residues positioned 17 to 19 bp and 26 to 31 bp fromthe ACS (38). Similarly, at the ARS1 B1 element, single nucleotides17 to 20 bp distal to the ACS are important for ARS activity(37). Finally, several studies have noted a bias toward AT basepairs within the B1 region at many ARS elements (6, 47, 51).

We showed, using multiple sequence alignment of active or phylogeneticallyconserved origins, that a WTW sequence is conserved within theB1 region at positions 17 to 19 bp from the ACS (P $≤$ 1 x 10^–10).We found no other DNA sequence conservation within multipleARS elements apart from the EACS and the WTW sequence; however,there was a small bias for AT-rich sequences 8 to 10 bp fromthe ACS (Fig. 6B). The WTW sequence is likely to be conservedat multiple replicators because it makes an important contributionto ARS activity. A double point mutation from WTW to WGG atARS313, ARS315, ARS316, and ARS318 caused a six- to eightfoldincrease in the plasmid loss rate, indicating that ARS functionhad been substantially impaired. A similar mutation within ARS319(a subtelomeric X-element ARS) caused a twofold increase inthe loss rate. The corresponding nucleotides at ARS307 are TTA,and mutation of this sequence to GGG impaired ORC binding invitro and impairs ARS activity in vivo (38). Furthermore, invitro ORC DNA footprinting studies showed that ORC contactsnucleotides overlapping this region (21). Thus, we infer thatmutations within the WTW sequence generally impair ORC bindingin vivo.

Mutation of the WTW sequence has different effects on ARS activitythat might depend on the strength of the ORC-DNA interactionat each particular origin. In support of this idea, mutationof the WTW motif within ARS317, which binds ORC 10-fold moretightly than at the efficient ARS1 origin (34), had no effecton ARS activity. One explanation for this finding is that additionalnucleotides within ARS317 besides EACS and WTW contribute totight ORC binding and, therefore, ORC binding is less affectedby mutations in the WTW sequence. In contrast, the principalstabilizing ORC-DNA contacts at ARS313 or ARS315 (for example)are perhaps mediated by the EACS and WTW alone.

We also performed a detailed linker scan analysis of the HMR-Isilencer origin ARS318. Although this ARS element has littlechromosomal replicator activity, it is important for transcriptionalsilencing at the HMR silent mating-type locus (reviewed in reference42). Deletion of HMR-I has a modest effect on HMR silencingin the wild-type strain but substantially impairs silencingwhen combined with mutations in HMR-E (1, 5, 40). Thus, a detailedanalysis of the functional components of this silencer elementprovide a starting point for further studies to test how thiselement cooperates to determine the epigenetic state of thesilencer. The first surprise was that a 9/11-bp match to theACS was required for origin activity and not the 10/11-bp match.This finding highlights the necessity of empirically determiningthe ACS at each ARS element, because these elements still cannotbe predicted accurately. Although ARS318 conforms to the A-B1-B2structure seen for several origins, it is unusual in that ithas an Afb1p binding site 6 bp 5' to the ACS. The Abf1p DNaseI footprint at this origin shows that Abf1p protects 21 nucleotidessurrounding the Abf1p site (9) (Fig. 2). ORC recognizes theACS at multiple origins and protects $~$ 10 to 12 bp 5' to the ACSfrom DNase I digestion (3, 14). ORC subunits can also be cross-linkedto DNA within this same interval (21). A DNase I footprint ofORC at HMR-I revealed a pattern resembling two ORC binding sites,with the stronger protection adjacent to and overlapping theAbf1p binding site (3). This agrees well with our molecularcharacterization of ARS318, indicating that the 9/11-bp matchadjacent to the Abf1p binding site is the essential ACS thatlikely binds ORC in vivo. The second region of DNase I protectionseen in vitro is consistent with an ORC-B2 interaction. Furthermore,the Abf1p and ORC proteins could conceivably contact each otherat ARS318 or they could bind to opposite faces of the DNA helix.

How does Abf1p enhance origin or silencer activities? AlthoughAbf1p stimulates silencing at HMR-E and HML-E, it is thoughtto do this by its ability to position nucleosomes flanking thesilencer and not through recruitment of silencer proteins (17).Abf1p has not been shown to make protein-protein contacts withknown heterochromatin proteins. Abf1p similarly stimulates ARS1origin activity through its ability to exclude nucleosomes fromthe B region, which is where prereplicative complex assemblyoccurs (23, 45). Interestingly, ORC has also been shown to positionnucleosomes adjacent to the silencer and at origins of replication(23). Since ORC can also position nucleosomes, it is unclearwhy Abf1p would stimulate origin activity at ARS318 two- tothreefold through a similar activity. We have now defined thefunctional cis elements at ARS318 for replicator activity, whichwill allow the contribution of the Abf1p and ORC binding sitesto be determined for transcriptional silencing. We propose thatthe ability of Abf1p to bend DNA (27) may alter ORC-DNA interactionsto stimulate ORC binding to the origin or, alternatively, stimulatesubsequent steps in origin activation. Abf1p-mediated DNA bendingmight also facilitate the activity of the silencer. In any event,defining the functional sequences of ARS318 allows further investigationof these sites to determine their precise roles in origin andsilencer function. The ARS318 replicator is inhibited by itschromosomal context since deleting HMR-E (which includes ARS317)results in substantial ARS318 replicator activity. Since ARS317is active in only $~$ 10% of cell cycles, it seems unlikely thatthe infrequent firing of this replicator inactivates ARS318.It is more likely that the heterochromatic environment at HMRinhibits ARS318 activity. This contrasts with the ARS301 replicatorat HML-E, which is also active in a plasmid context but inactiveon the chromosome, since the loss of transcriptional silencingdoes not lead to its activation (16). Therefore, other positionaleffects likely inhibit ARS301.

ACKNOWLEDGMENTS

We thank Catherine Fox for helpful discussions, Ryan Frischand Don Pappas for several plasmids, and Craig Webb for supportingJ.M. in the Laboratory of Metastasis and Angiogenesis at theVan Andel Research Institute.

C.S.N. and J.F.T. were supported by NIH grant GM35679, and M.W.was supported by the Van Andel Research Institute.

FOOTNOTES

* Corresponding author. Mailing address: Laboratory of Chromosome Replication, Van Andel Research Institute, Grand Rapids, MI 49503. Phone: (616) 234-5306. Fax: (616) 234-5307. E-mail: michael.weinreich{at}vai.org

Published ahead of print on 23 June 2008.

Supplemental material for this article may be found at http://mcb.asm.org/.

REFERENCES

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