Linear Derivatives of Saccharomyces cerevisiae Chromosome III Can Be Maintained in the Absence of Autonomously Replicating Sequence Elements

Replication origins in Saccharomyces cerevisiae are spaced atintervals of approximately 40 kb. However, both measurementsof replication fork rate and studies of hypomorphic allelesof genes encoding replication initiation proteins suggest thequestion of whether replication origins are more closely spacedthan should be required. We approached this question by systematicallydeleting replicators from chromosome III. The first significantincrease in loss rate detected for the 315-kb full-length chromosomeoccurred only after all five efficient chromosomal replicatorsin the left two-thirds of the chromosome (ARS305, ARS306, ARS307,ARS309, and ARS310) had been deleted. The removal of the inefficientreplicator ARS308 from this originless region caused littleor no additional increase in loss rate. Chromosome fragmentationsthat removed the normally inactive replicators on the left endof the chromosome or the replicators distal to ARS310 on theright arm showed that both groups of replicators contributesignificantly to the maintenance of the originless chromosome.Surprisingly, a 142-kb derivative of chromosome III, lackingall sequences that function as autonomously replicating sequenceelements in plasmids, replicated and segregated properly 97%of the time. Both the replication initiation protein ORC andtelomeres or a linear topology were required for the maintenanceof chromosome fragments lacking replicators.

INTRODUCTION

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INTRODUCTION

In eukaryotes, DNA replication initiates at specific sites calledreplication origins. cis-acting sequences called replicatorsdefine the positions and regulate the activity of replicationorigins by promoting the assembly of prereplicative complexes(pre-RCs) during the G₁ phase of the cell cycle. Eukaryoticreplicators were first identified in the budding yeast Saccharomycescerevisiae on the basis of their ability to promote the extrachromosomalmaintenance of plasmids (22, 50). The dissection of these autonomouslyreplicating sequence (ARS) elements revealed an essential 11-bpsequence, called the ARS consensus sequence (ACS), that is requiredfor both plasmid and chromosomal replicator activity (reviewedin reference 34). The ACS is the core of the binding site forthe highly conserved six-subunit origin recognition complex(ORC), which recruits and assembles the other components ofthe pre-RC (reviewed in reference 1). Like the ORC, the othercomponents of the pre-RC are highly conserved throughout theeukaryotic kingdom.

Upon entry into S phase, pre-RCs are activated to initiate replicationaccording to a temporal program whose determinants are poorlyunderstood. The activation of replication origins requires theactivity of two kinases, a cyclin-dependent kinase composedof the catalytic subunit encoded by CDC28 and regulatory subunitsencoded by CLB5 and CLB6, and the Dbf4-dependent kinase, composedof a catalytic subunit encoded by CDC7 and a regulatory subunitencoded by DBF4. During replication initiation, DNA is unwoundat origins and replication fork proteins are assembled to formreplisomes, which move bidirectionally away from origins (reviewedin reference 2).

ARS elements and replication origins have been mapped in theyeast genome by a variety of approaches. In early studies, ARSelements were identified on chromosomes III and VI by screeningoverlapping fragments of the chromosomes for ARS activity inthe plasmid assay (37, 41, 48). ARS elements were then examinedfor chromosomal replication origin activity by two-dimensionalgel electrophoresis of replication intermediates (9, 15, 18,20, 23, 24, 41, 55, 56, 67). These studies demonstrated thatARS elements are required for replication origin activity andthat not all ARS elements are detectably active as chromosomalreplication origins. More recent studies have used genome arraysto identify replication origins. The first approach made useof density transfer experiments to generate a genome-wide replicationtiming profile (43). Replication origins could be identifiedas regions that replicated earlier than their neighboring sequences.The second approach was based on the twofold increase in copynumber caused by replication (66). Again, regions that doubledin copy number before their neighboring sequences were consideredto be replication origins. These timing profiles, which wouldnot detect inefficient replication origins, identified 332 and247 replication origins, respectively, suggesting an averagespacing of 40 kb between replication origins in the 12-Mb yeastgenome. This average spacing is consistent with spacing deducedin early electron microscopy and fiber autoradiography studies(35, 44). A third approach used genome arrays to identify thebinding sites of two pre-RC components, ORC and the Mcm2-7 complex,by chromatin immunoprecipitation (64). This approach, whichhas the potential to identify both efficient and inefficientreplication origins, identified 429 potential origins in theyeast genome.

Several lines of evidence suggest that replication origins aremore closely spaced than should be required. For example, intheir genome-wide study of replication dynamics, Raghuramanet al. (43) found that replication forks move at a mean rateof 2.9 kb/min during an S phase of approximately 55 min. Thus,forks from a single bidirectional replication origin that initiatesearly in S phase should be able to replicate about 320 kb ofDNA. Although this calculation does not take into account thepresence of replication fork barriers or pause sites, whichare known to be present in the yeast genome (5, 10, 19), itsuggests that replication origins might be redundant. Consistentwith this idea, hypomorphic alleles of genes encoding componentsof the pre-RC reduce the efficiency of replication origin firing(16, 27) and therefore increase the spacing between replicationorigins without affecting cell viability. Moreover, a hypomorphicallele of the essential Cdc7p protein kinase, whose activityis required for replication initiation, also causes a reductionin the efficiency of replication origin firing (13), and thedeletion of CLB5, one of the two S-phase cyclins, prevents latereplication origins from firing (14). Although the length ofS phase was extended in each of the replication mutants, theseresults suggest that a genome-wide increase in origin spacingis tolerated. However, the extent of the increase in originspacing was not determined in these studies.

Because of the detailed mapping of ARS elements and replicationorigins on chromosome III, we undertook a study of the effectsof deleting replication origins on chromosome stability. Inan early study, we found that deleting either of the two efficientreplicators, ARS307 and ARS309, from a small circular derivativeof chromosome III caused an increase in its loss rate and thecircular chromosome could not be maintained in the absence ofboth ARS307 and ARS309; however, deleting both of these efficientreplication origins from the full-length chromosome III hadno effect on its loss rate (8). To further examine the effectof deleting replication origins, we continued our analysis ofthe full-length chromosome. The first significant increase inloss rate detected for the full-length chromosome occurred onlyafter all five efficient chromosomal replicators between theleft end of the chromosome and the MAT locus were deleted. Theremoval of the inefficient replicator ARS308 from this originlessregion caused little or no additional increase in loss rate.Chromosome fragmentations which removed the normally inactivereplicators on the left end of the chromosome and/or the replicatorsdistal to MAT on the right arm showed that both groups of replicatorscontributed significantly to the maintenance of this chromosome.Surprisingly, a 142-kb derivative of chromosome III, lackingall sequences that function as ARS elements in plasmids, replicatedand segregated properly 97% of the time. Both the replicationinitiator protein ORC and telomeres or a linear topology wererequired for maintenance of chromosome fragments lacking efficientreplicators.

MATERIALS AND METHODS

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MATERIALS AND METHODS

Strains. Escherichia coli strains JA226 and DH5 ${alpha}$ were used for plasmidDNA preparations (56). Yeast strains are listed in Table 1.Strain CF4-16B33 was used for the construction of all ARS deletionsand chromosome fragmentations as described below. Strains witha balancer chromosome III derived from Saccharomyces carlsbergensiswere made by crossing CN5-19a with the disomic MATa/MAT ${alpha}$ strainsCF4-16B33 and YDN292 and isolating rare prototrophic diploidstrisomic for chromosome III. These strains were sporulated andHis⁺ Ade⁺ Ura^– Trp^– MATa spores that segregatedred colonies that were His^– Ade^– Ura^– Trp^–were identified, yielding strains CB08D and YDN288. The S. cerevisiaechromosome III in these strains was subsequently fragmentedto the right of ARS310. The orc2-1 and orc5-1 mutations wereintroduced into strain YKN6 by the two-step gene replacementprocedure (3), yielding strains YNS1 and YNS3. Plasmids carryingthe orc2-1 and orc5-1 mutations were provided by A. Dillin andJ. Rine (University of California, Berkeley). A suppressor mutationthat allows some orc2-1 strains to grow at 30°C has beenreported (47). The orc2-1 strains used in this study failedto grow at 30°C.

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TABLE 1. Yeast strains

Strains carrying the 61-kb ring chromosome marked with SUP11-1(8) were made by crossing the kar1- ${Delta}$ 15 strain YMS499-1 with strainsPF1-7D (carrying the 0ORI ${Delta}$ ring) and PF6-5B (carrying the ARS309 ${Delta}$ ring) (8) and identifying Leu⁺ Can^r MATa segregants in whichthe ring had been transferred to the YMS499-1 nucleus by chromoduction(25). Additional ARS deletions were made in the ring as describedbelow. Ring chromosomes carrying internal telomere repeat sequences(TRS) were made using plasmid pYND125 as described below. TheSUP11-1-marked linear 61-kb chromosome fragments were made byfirst constructing a linear derivative of the 61-kb ring carryingthe ARS307 ${Delta}$ in strain 33-4 (8) by using plasmid pNO8. SUP11-1was then integrated near the ARS307 ${Delta}$ by using C2G::SUP11-1 (8).Additional ARS deletions were introduced as described below.

Plasmids. For the deletion of ARS elements, we constructed plasmids carryingARS deletions ranging in size from 0.25 kb to 3.2 kb in theURA3 vectors YIp5 (51) or pRS306 (49) for use in two-step genereplacements (3). The sequences deleted corresponded to thesmallest subclones with ARS activity that we had identified(37). Each of the replicator deletion constructs was demonstratedto lack ARS activity, and substitution of the chromosomal copyof the ARS element with the deletion caused the abrogation ofchromosomal replicator activity (8, 9). Plasmids for the deletionof ARS307 and ARS309 were described by Dershowitz and Newlon(8), and the plasmid for deletion of ARS306 was described byDeshpande and Newlon (9). The construction of other ARS deletionplasmids is described in the supplemental material. The ARS305deletion removes a 1.1-kb BamHI-ClaI ARS305 fragment extendingfrom positions 38606 to 39706 of the chromosome III sequence(www.yeastgenome.org). The ARS310 deletion removes either a3.4-kb KpnI fragment, creating a deletion extending from positions164426 to 167613 of the chromosome III sequence, or an 846-kbEcoRV fragment extending from positions 166499 to 167344 ofthe chromosome III sequence (www.yeastgenome.org). ARS308 wasremoved by replacing CEN3 with CEN4.

To construct plasmids for chromosome fragmentation (62), weinserted a 340-bp C₄A₂ Tetrahymena telomere fragment in theintegrating vectors pRS304 and pRS306 (49) to serve as a seedfor S. cerevisiae telomere addition, following linearizationof the plasmid by restriction enzyme digestion. Chromosome IIIsequences were inserted in the polylinker on the other sideof the restriction enzyme site to target chromosome fragmentations.Plasmids designed to fragment the right arm at two sites, position117326, approximately 2.4 kb to the right of CEN3 (pYND97),and positions 174361, 5 kb to the right of ARS310 (pYND56),were made in pRS304. Plasmids designed to fragment the chromosomeat three positions on the left arm were made in pRS306. pYND103fragments the chromosome at position 6582, approximately 5 kbto the left of ARS301. pYND101 fragments the chromosome at position17033, approximately 2 kb to the right of ARS320. pYND104 fragmentsthe chromosome at position 31871, approximately 1.5 kb to theright of ARS304.

Plasmid pNO8 was used to linearize the 61-kb ring chromosomenear ARS309. Plasmid pYND125 was used to insert telomere repeatsequences into the 61-kb ring chromosome and its ARS deletionderivatives using a two-step gene replacement.

Loss rate measurements. Chromosome loss rates were measured by fluctuation analysisusing the colony isolation method previously described (8).To distinguish chromosome loss from mitotic recombination orgene conversion events that caused the loss of SUP11-1, redcolonies were tested for histidine prototrophy and their abilityto mate. Chromosome loss events produced His^– MATa colonies.White and red colonies from the plates used to isolate coloniesfor loss rate determinations were tested for the presence ofthe expected ARS deletions by Southern analysis.

Analysis of replication intermediates. Methods for DNA isolation, two-dimensional gel electrophoresis,and fork direction analysis were described previously (56).

Chromoductions. Chromoductions (25) were performed by mating a kar1- ${Delta}$ 15 (58)recipient strain with a strain carrying the chromosome III derivativeto be transferred. Chromoductants were selected on plates containing60 µg/ml canavanine and 10 µg/ml cycloheximide butlacking arginine, leucine, and tryptophan.

RESULTS

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RESULTS

Deletion of active replicators causes only modest chromosome instability. To examine the dependence of chromosome stability on the presenceof replicators, we systematically deleted replicators from theleftmost 55% of chromosome III and measured the effects on therates of chromosome loss by fluctuation analysis (8). For theseexperiments, we made use of a haploid ade2-101 strain that wasdisomic for chromosome III. The two copies of chromosome IIIcarry complementing his4 alleles and are heterozygous for matingtype. The chromosome from which replicators were deleted carriedan insertion of the ochre suppressor tRNA, SUP11-1, 7.8 kb tothe left of CEN3. Therefore, the disomic strain is a His⁺ nonmaterthat forms white colonies as a result of the suppression ofade2-101 by SUP11-1. Loss of the test chromosome is signaledby a red colony or a red sector that is His^– Ade^–MATa.

Figure 1A shows the locations and relative efficiencies of replicatorsalong chromosome III. In most cells, this chromosome is replicatedby forks that initiate at the seven efficiently used replicators:ARS305, ARS306, ARS307, ARS309, ARS310, ARS315, and ARS319 (36,41). The removal of up to four of the five efficient replicatorsbetween the left telomere and MAT had no effect on the lossrate. Each of the replicator deletions was studied individuallyand in combination with other deletions (Fig. 1B). Two-tailedt tests, analysis of variance tests, and nonparametric analogswere used to assess the statistical significance of differencesin loss rates. The effect of deleting ARS306 and ARS307 (datanot shown), which are both on the left arm, was not significantlydifferent from that of deleting ARS307 and ARS309, which flankthe centromere (8). These pairs of deletions created regionsof approximately 100 kb that lack efficient replicators. Similarly,deleting ARS305, ARS306, and ARS307, which removed all efficientchromosomal replicators from the left arm, or ARS307, ARS309,and ARS310 yielded chromosomes with wild-type loss rates. Chromosomesfrom which four ARS elements, either ARS305, ARS306, ARS307,and ARS309 or ARS306, ARS307, ARS309, and ARS310 or ARS307,ARS308, ARS309, and ARS310, were deleted also showed wild-typeloss rates. It was not until all five efficient replicatorsin the 194-kb region between the left telomere and MAT (ARS305,ARS306, ARS307, ARS309, and ARS310) were deleted to producethe 5ORI ${Delta}$ chromosome that there was a modest two- to threefoldincrease in the loss rate. ARS308 is tightly associated withCEN3 (20). Replacing CEN3 with CEN4, which does not have anassociated ARS element, created the 6ORI ${Delta}$ chromosome and causedno further statistically significant increase in loss rate.The loss rates for chromosomes with zero to four ARS deletionswere significantly lower that the loss rates for chromosomeswith five or six ARS deletions (P = 0.002). Thus, the 164-kbregion of the chromosome between ARS304 and ARS313 can be replicatedby forks that initiate outside the region. Because ARS304 andthe ARS elements to its left, as well as ARS314, are not detectablyactive in wild-type strains and ARS313 is only weakly active,these results raise the possibility that the entire 225-kb regionto the left of ARS315, which comprises 71% of the chromosome,can be replicated by a fork originating at ARS315.

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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.

A chromosome III derivative lacking all ARS elements is surprisingly stable. To assess the role of the remaining replicators in maintainingthe chromosome, we performed a series of chromosome fragmentations(62) to remove the remaining ARS elements (Fig. 2). The fragmentationof chromosome III just to the right of ARS304, which produceda 283-kb chromosome fragment, did not affect the stability ofthe chromosome carrying no ARS deletions (Fig. 2). However,the loss rate of the 283-kb 6ORI ${Delta}$ chromosome, lacking ARS305through ARS310, increased approximately 37-fold, suggestingthat one or more of the six normally inactive replicators onthe left end of the chromosome contributes to the replication6ORI ${Delta}$ of the chromosome. Consistent with this idea, in the 6ORI ${Delta}$ strain we found very weak bubble arcs associated with ARS301and ARS303 in two-dimensional gel patterns of replication intermediates(data not shown), confirming a previous report that these twoARS elements became active replicators in a chromosome fromwhich ARS305 and ARS306 were deleted (63). Replication intermediatesof both the balancer chromosome and the 6ORI ${Delta}$ chromosome wereseen in these two-dimensional gel patterns. Therefore, if ARS301and ARS303 were fully active in the 6ORI ${Delta}$ chromosome, the intensitiesof the bubble arcs and Y arcs should have been similar. However,the bubble arcs were much less intense than the Y arcs, suggestingthat these replicators function inefficiently in the 6ORI ${Delta}$ chromosome.

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FIG. 2. Loss rates of chromosomes fragmented near ARS304, ARS310, or both. Diagrams on the left show the 0ORI ${Delta}$ derivatives of chromosome III used. 0ORI ${Delta}$ , 5ORI ${Delta}$ , and 6ORI ${Delta}$ 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.

We also examined the stability of 174-kb chromosomes fragmented6.9 kb to the right of ARS310 to remove the remaining sevenreplicators on the right arm (Fig. 2). The results were similarto those found for the fragmentations near ARS304, with no effecton the 0ORI ${Delta}$ control and a 30-fold increase in the loss rateof the 6ORI ${Delta}$ chromosome (Fig. 2). These results demonstrate thatreplicators distal to ARS310 on the right arm also contributeto the replication of the 6ORI ${Delta}$ chromosome.

Finally, we fragmented the 0ORI ${Delta}$ and 6ORI ${Delta}$ chromosomes to theright of ARS304 and to the right of ARS310, producing a 142-kbchromosome III fragment. While the stability of the 0ORI ${Delta}$ chromosomefragment was unaffected, the loss rate of the 6ORI ${Delta}$ chromosomefragment increased by more than 200-fold relative to the full-length6ORI ${Delta}$ chromosome (Fig. 2). Nevertheless, it is remarkable thatthe 6ORI ${Delta}$ doubly fragmented chromosome, which carries no sequencethat functions as an ARS element in the plasmid assay, stillreplicates and segregates correctly in 97% of cell divisions.

Because we had seen no significant difference in the loss ratesof the full-length 5ORI ${Delta}$ and 6ORI ${Delta}$ chromosomes, we also examinedthe loss rates of the fragmented 5ORI ${Delta}$ chromosomes, which retainthe CEN3-associated inefficient replicator ARS308 (Fig. 2).While the loss rates of the 5ORI ${Delta}$ fragments were not significantlydifferent from the loss rates of the 6ORI ${Delta}$ fragments, each ofthe 6ORI ${Delta}$ loss rates was higher than the corresponding 5ORI ${Delta}$ lossrate, suggesting that ARS308 contributes in a minor way to themaintenance of the 5ORI ${Delta}$ fragments.

One explanation for the unexpectedly high stabilities of origindeletion chromosomes is that they regained one or more originsby gene conversion or reciprocal recombination with the balancerchromosome. To examine this possibility, we used Southern analysisto test for the presence of ARS deletions and, where appropriate,the presence of the new telomere(s) in both white disomic coloniesand red colonies that had lost the SUP11-1-marked chromosome.Colonies from each fluctuation experiment and from the strainscreated for analysis of chromosome stability were analyzed.Events that restored replication origins were rare. Only 1 of58 5ORI ${Delta}$ or 6ORI ${Delta}$ full-length chromosomes regained an ARS element,and only 4 of 50 singly fragmented 5ORI ${Delta}$ or 6ORI ${Delta}$ chromosomesregained one or more ARS elements by gene conversion or reciprocalrecombination. We also deleted RAD52, which is required forall homologous recombination. rad52 mutants are known to exhibitincreased chromosome loss rates (21, 33). We found that theloss rate of the 0ORI ${Delta}$ fragment was increased to the same extentas the loss rate of the 5ORI ${Delta}$ fragment (data not shown). Therefore,the maintenance of these origin deletion chromosomes does notdepend on the acquisition of a replication origin.

Further evidence that homologous interaction with the balancerchromosome did not contribute substantially to the maintenanceof the 5ORI ${Delta}$ or 6ORI ${Delta}$ chromosome was provided by the analysisof strains carrying a homeologous balancer chromosome derivedfrom the alloploid brewing yeast S. carlsbergensis (57, 68)in place of the S. cerevisiae balancer. This brewing yeast chromosome,which can substitute for S. cerevisiae chromosome III function,is a mosaic in which the sequences between the left telomereand MAT were derived from a chromosome diverged in DNA sequencefrom the S. cerevisiae homeologue, and the sequences distalto the MAT locus on the right arm are S. cerevisiae-like (reviewedin reference 26). Mitotic recombination rates between sequenceson this chromosome and S. cerevisiae sequences are reduced 10-to 20-fold in the homeologous regions (42). If homologous interactionswere important for the maintenance of the 5ORI ${Delta}$ or 6ORI ${Delta}$ chromosome,then their stabilities should be reduced in strains carryingthe S. carlsbergensis balancer. Measurements of loss rates ofS. cerevisiae chromosome III in this strain background revealedthat the loss rates of the 315-kb 0ORI ${Delta}$ full-length chromosome([3.7 ± 1.8] x 10^–5 loss/division) and 174-kb 0ORI ${Delta}$ chromosome fragmented at ARS310 ([7.0 ± 2.8] x 10^–5loss/division) were not significantly different from the lossrates measured in strains with the S. cerevisiae balancer (Fig.2). The 5ORI ${Delta}$ and 6ORI ${Delta}$ full-length chromosomes ([3.0 ±1.0] x 10^–4 and [4.4 ± 1.0] x 10^–4 loss/division,respectively) and the 174-kb 5ORI ${Delta}$ chromosome fragmented nearthe position of ARS310 ([2.4 ± 0.5] x 10^–3 loss/division)were lost at rates approximately twofold higher in these strainsthan in strains with the S. cerevisiae balancer (Fig. 2). The174-kb 6ORI ${Delta}$ chromosome fragmented near the position of ARS310([1.1 ± 0.3] x 10^–2 loss/division) was lost ata rate approximately fivefold higher in these strains. Thesemodest increases in loss rate of the 5ORI ${Delta}$ and 6ORI ${Delta}$ chromosomesfurther indicate that homology with the balancer chromosomeis not a major factor in the maintenance of the originless chromosomes,although we cannot exclude the possibility of a role for shortregions of homology that are not able to direct efficient homologousrecombination.

Replication of the 6ORI ${Delta}$ fragment. To further explore the role of the six inactive ARS elementsin the maintenance of the 6ORI ${Delta}$ chromosome fragmented to theright of the position of ARS310, we undertook an additionaltwo-dimensional analysis of replication intermediates and constructedadditional fragmentations at both ends. For the two-dimensionalgel analysis, we used a strain carrying the homeologous S. carlsbergensischromosome III balancer, which made it possible to find probesthat hybridize only to the S. cerevisiae chromosome at highstringency, allowing the visualization of replication intermediatesthat arise specifically from the 6ORI ${Delta}$ fragment. Analyses ofreplication intermediates of ARS301, ARS302, ARS303/320, andARS304 are shown in Fig. 3. Bubble arcs are present in the ARS301and ARS303/320 patterns of the 174-kb 6ORI ${Delta}$ fragment, but notin the 174-kb 0ORI ${Delta}$ fragment. We have never observed origin activityof ARS302 or ARS304 in its normal chromosomal context.

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FIG. 3. Two-dimensional gel analysis of replication intermediates of ARS301, ARS302, ARS303/ARS320, and ARS304 in 0ORI ${Delta}$ and 6ORI ${Delta}$ chromosomes fragmented near ARS310. DNA was prepared from strains CB08D3 (0ORI ${Delta}$ ) and YDN289 (6ORI ${Delta}$ ), 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 0ORI ${Delta}$ chromosome fragment and the right panel shows the pattern obtained from the 6ORI ${Delta}$ fragment. A diagram of the 174-kb 6ORI ${Delta}$ chromosome fragment examined is shown at the bottom of the figure. Landmarks on the map line are labeled as described for Fig. 2.

Results of stability measurements on a series of 174-kb 5ORI ${Delta}$ chromosomes carrying further fragmentations at the left endin strain F510 corroborated the role of the ARS elements flankingHML in the maintenance of the 174-kb 5ORI ${Delta}$ fragment. Removing6.5 kb of the left end of the chromosome, including ARS300,caused no increase in the loss rate of this fragment ([1.3 ±0.3] x 10^–3 loss/division) relative to the 174-kb chromosomefragment (Fig. 2). In contrast, the removal of 17 kb of theleft end of the chromosome, including HML as well as ARS301,ARS302, and ARS303/ARS320, caused an increase in loss rate equivalentto that seen for the 142-kb 5ORI ${Delta}$ fragment ([1.8 ± 0.6]x 10^–2 loss/division) (Fig. 2). Therefore, of the sixinactive ARS elements on the left end of chromosome III, itappears that replication initiations at only ARS301 and ARS303/ARS320contribute significantly to the maintenance of the 174-kb 5ORI ${Delta}$ chromosome III fragment.

If the 6ORI ${Delta}$ fragment were replicated by initiations at ARS301and ARS303/320, then replication forks should diverge from thesetwo ARS elements. To test this hypothesis and to gain insightinto how the remainder of the fragment is replicated, we determinedthe direction of fork movement along the 174-kb 6ORI ${Delta}$ chromosomeby using a modification of the two-dimensional gel procedure(17). Figure 4 shows the analysis of fork movement through fragmentsimmediately to the left of HML, ARS305, ARS307, and ARS310.In the 174-kb 0ORI ${Delta}$ control fragment, the patterns showed onlyforks moving to the left from the nearest origin, except nearARS310. The fainter fork moving rightward into ARS310 demonstratesthat ARS310 initiates replication in approximately 70% of cellcycles, confirming our previous observations in another strain(56).

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FIG. 4. Analysis of the direction of replication fork movement along the 0ORI ${Delta}$ and 6ORI ${Delta}$ 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 0ORI ${Delta}$ fragment, the middle panel shows the pattern obtained from the 6ORI ${Delta}$ 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 6ORI ${Delta}$ 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.

The presence of diverging forks in regions flanking HML in the174-kb 6ORI ${Delta}$ fragment provides further support for the idea thatreplication initiates at latent origins surrounding HML. Sincewe detected only forks moving to the left from HML in the ARS301panel but detected forks moving in both directions at approximatelyequal frequency to the right of HML in the ARS305 panel, weconclude that replication initiates at ARS301 and/or ARS303only about half of the time.

We also detected forks moving in both directions just to theleft of the ARS307 and ARS310 deletions in the 6ORI ${Delta}$ fragment(Fig. 4). It is particularly striking that at least 30% of theforks that replicate the region adjacent to the ARS310 deletionmove leftward through the fragment, indicating that they musthave initiated distal to the ARS310 deletion. The new telomereseeded by a C₄A₂ tract in the fragmentation vector is locatedonly 6.8 kb to the right of the ARS310 deletion, suggestingeither that there is a replication origin in this 6.8 kb ofchromosome III or that replication is initiating at the newtelomere. The fragmentation vector lacks ARS activity, makingit unlikely that replication initiates in vector sequences.

A potential mechanism for the introduction of a replicator inthis region is the acquisition by the new telomere of a subtelomericX or Y' ARS element from another chromosome. The presence ofan X element would extend the length of the new telomere byapproximately 500 bp, and a Y' element would add 5 to 6.2 kbof sequence with a characteristic restriction pattern (7). Thenew telomere was examined by Southern analysis, making use ofan EcoRV site in the fragmentation vector and a plasmid probe,to determine the size of the new telomere. The somewhat diffuseband revealed, centered at about 1.6 kb, was predicted by theexpected addition of C_1-3A repeats to the C₄A₂ tract of thefragmentation vector by yeast telomerase (54) and is inconsistentwith the presence of either a subtelomeric X or a subtelomericY' element (data not shown).

To test whether specific sequences on the right arm of the 174-kb5ORI ${Delta}$ fragment contribute to its maintenance, we fragmented thischromosome 2.4 kb to the right of CEN3. The loss rate of theresulting 117-kb fragment ([9.7 ± 0.4 ] x 10^–4loss per division) was not different from the loss rate of the174-kb 5ORI ${Delta}$ or 6ORI ${Delta}$ fragment (Fig. 2), making it unlikely thatthere is an additional replication origin on the right arm ofthe 174-kb fragment that contributes to its maintenance.

Taken together, these results are consistent with at least twomechanisms for the maintenance of the chromosome III fragmentlacking conventional replication origins. First, in any givenchromosome fragment, replication could initiate at one of severalreplication origins that are so inefficient that they cannotsupport the maintenance of a plasmid in the ARS assay. Second,replication could initiate by an unknown mechanism at one endor the other of the fragment, but not at both ends in most molecules.Both models would account for the demonstrated bidirectionalmovement of replication forks at several different places alongthe 174-kb 6ORI ${Delta}$ fragment.

Linear topology or telomeres are required for the maintenance of a chromosome fragment lacking ARS elements. We showed previously that a 61-kb ring chromosome (Fig. 5A)from which the two efficient replication origins, ARS307 andARS309, were removed could not be stably maintained; at veryhigh frequency, one of the two replication origins was restoredby gene conversion (8). To test whether this fragment of chromosomeIII, which was produced by a recombination between a Ty2 tothe left of LEU2 and a Ty1 to the right of PGK1, could be maintainedas a linear fragment, we introduced a URA3 gene flanked by C₄A₂tracts near ARS309 and selected for linear derivatives of thering chromosome on plates containing 5-fluoroorotic acid (32).The presence of the expected new telomeres on the 61-kb linearfragment (Fig. 5C) was confirmed by Southern blotting (datanot shown). Replicators were then deleted as described above.Consistent with our previous results (8), the deletion of eitherARS307 or ARS309 from the 61-kb ring chromosome caused a three-to fourfold increase in loss rate and a derivative lacking bothefficient replicators was not recovered (Fig. 5; Table 2). Incontrast, the 2ORI ${Delta}$ linear derivatives from which the efficientreplicators ARS307 and ARS309 were deleted or the 3ORI ${Delta}$ linearderivative lacking all three replicators were maintained andshowed only two- or threefold increases in loss rate relativeto the linear derivative with only ARS307 deleted. The lossrate of the linear carrying the ARS307 deletion was similarto the loss rate of the ring from which it was derived.

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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.

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TABLE 2. Comparison of stabilities of 61-kb circular and linear derivatives of chromosome III

The 61-kb ring chromosome and its linear derivative containidentical DNA sequences except for the telomeres on the linearform. Longtine et al. (30) have reported that TRSs stabilizesmall ARS plasmids by improving their ability to segregate properly.We undertook two experiments to test the idea that the presenceof a TRS would improve the stability of the 61-kb ring chromosome.First, we made use of the SUP11-1 marker on the ring chromosometo determine whether the loss events caused by the deletionof replication origins from the 61-kb ring chromosome and itslinear derivatives were due to failure to replicate (1:0 lossevents) or failure to segregate (2:0 loss events). The straincarrying a single copy of the 61-kb ring gives rise to coloniesthat are slightly pink on plates with limiting adenine, dueto the incomplete suppression of the ade2-101 allele in thestrain, while strains carrying two copies of SUP11-1 give riseto white colonies. Moreover, the sectoring patterns of the coloniesare different, with colonies produced by cells with a singlecopy of the SUP11-1-marked fragment frequently having multiplered sectors, while colonies produced by cells carrying two copiesof the marked chromosome fragment infrequently having sectorsbecause the production of a red sector requires independentlosses of both marked fragments. Therefore, missegregation eventsare expected to cause an accumulation of white, nonsectoringcolonies along with red colonies that lack the fragment, whilefailures to replicate are expected to give rise to pinkish sectoringcolonies along with red colonies. The number of cells platedfor fluctuation analysis was optimized to determine the rateof production of loss events (signaled by red colonies), whichinclude both 1:0 and 2:0 segregations. Nevertheless, it waspossible to estimate that the rate of production of white, nonsectoringcolonies was (4.1 ± 2.3)x 10^–4, a rate 10-foldlower than the rate of loss events. Because reversions of theade2-101 mutation and new mutations in ADE1 both result in theproduction of white, nonsectoring colonies, the 10-fold-lowerrate of missegregation represents an minimum estimate of thedifference. Therefore, simple 1:0 loss events, presumably fromfailures to replicate, rather than missegregations, constitutethe predominant mechanism of loss of the 61-kb ring chromosome.

The second experiment was to insert a 276-bp TRS sequence into0ORI ${Delta}$ and 1ORI ${Delta}$ 61-kb ring chromosomes at the site that was usedto linearize the ring (Fig. 5 B) and to measure loss rates ofresulting chromosomes (Table 2). The loss rates of all threering derivatives carrying the TRS were 1.5- to 2-fold higherthan the loss rates of the corresponding rings lacking the TRS.Although the differences in loss rate were not significant intwo of the cases, these results indicate that the addition ofa TRS to the ring chromosomes does not improve their stability.Using the single ARS deletion constructs carrying the TRS, wealso attempted to delete the second efficient origin in thepresence of the TRS. Both the ARS307 and ARS309 deletion constructsnormally yield more ARS deletion popouts than wild-type popoutsin the two-step gene replacement procedure, but in this case,we failed to recover the 2ORI ${Delta}$ chromosome. We analyzed a totalof 26 Ura^– Leu⁺ popouts of single ARS deletion rings carryingTRS and found that 19 carried the wild-type copy of the ARSelement we were trying to delete. Two of the recovered ringchromosomes carried the wild-type copies of both ARS307 andARS309, and one carried the deletion we were trying to makebut had lost the first deletion. These three-ring chromosomesalmost certainly acquired wild-type ARS elements by gene conversionfrom the balancer chromosome. Finally, we found four very smallcolonies that grew on plates lacking Leu and containing 5-fluorooroticacid that were used to select Ura^– popouts. These smallcolonies were Leu^– and lacked the ring chromosome whenrestreaked. It is possible that these four colonies initiallycarried the 2ORI ${Delta}$ ring, but it was so unstable that it was lostin subsequent restreaking. These results indicate that internaltelomere repeat sequences are insufficient to stabilize the2ORI ${Delta}$ 61-kb ring chromosome and that the presence of telomererepeat sequences alone cannot explain the maintenance of thelinear chromosome fragment lacking replicators. Instead, itis likely that either the linear topology or the presence oftelomeres on the linear fragment supports replication initiationevents that cannot occur on the circular chromosome.

ORC is required for the maintenance of five-deletion fragments. The possibility that replication initiates at or near telomereson chromosome fragments lacking ARS elements raises the questionof whether the normal replication initiation machinery is requiredfor maintenance of these origin-deficient chromosomes. Therefore,we tested whether mutations in the replication initiator proteinORC affected the stability of chromosome fragments from whichefficient replicators have been deleted. Temperature-sensitivemutations in two subunits of ORC, orc2-1 and orc5-1, cause defectsin replication initiation at the permissive temperature, measuredboth by plasmid stability assays and by two-dimensional gelanalysis of chromosomal replication origins (12, 16, 27), althoughthe orc5-1 defect is more apparent at semipermissive temperatures.We introduced the orc2-1 and orc5-1 mutations into strain YKN6by two-step gene replacements. Consistent with published results,both mutant strains had a plasmid maintenance defect relativeto the wild-type strain, measured as the fraction of plasmid-bearingcells in cultures grown under selection for the plasmid (61).The ARSH4 CEN6 plasmid pRS315 (49) was present in 92.3% ±7.5% of YKN6 cells, 37.0% ± 2.2% of orc2-1 cells, and52.6% ± 8.3% of orc5-1 cells.

We then introduced both 174-kb and 142-kb 0ORI ${Delta}$ and 5ORI ${Delta}$ chromosomefragments into the strains by chromoduction and measured fragmentstabilities by fluctuation analysis (Fig. 6). Consistent withthe role of ORC in the activation of both efficient and dormantreplication origins, the orc2-1 mutation caused a significantincrease in the loss rate of the 174-kb 0ORI ${Delta}$ and 5ORI ${Delta}$ chromosomefragments and the 142-kb 0ORI ${Delta}$ chromosome fragment. Strikingly,we were unable to recover chromoductants carrying the 142-kb5ORI ${Delta}$ chromosome fragment. In all chromoductants analyzed bySouthern blotting, the 142-kb 5ORI ${Delta}$ fragment had acquired replicators,either by gene conversion of one or more replicators from thebalancer chromosome or by events that restored the right orleft end of the chromosome, resulting in the loss of one ofthe telomeres introduced by fragmentation. At the permissivetemperature (23°C), the orc5-1 mutation had no effect onthe loss rate of either the 174-kb or the 142-kb 0ORI ${Delta}$ or 5ORI ${Delta}$ chromosome fragments (data not shown). At the semipermissivetemperature of 27°C, the 142-kb 5ORI ${Delta}$ chromosome fragment,but not the other three fragments tested, was lost at a rate2.5-fold higher in the orc5-1 strain than in the wild-type strain(Fig. 6). Taken together, these results indicate that ORC isrequired for maintenance of the chromosome III fragment lackingARS elements.

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FIG. 6. Stabilities of 0ORI ${Delta}$ and 5ORI ${Delta}$ chromosomes fragmented near ARS310 or near ARS304 and ARS310 in orc5-1 and orc2-1 strains. Diagrams of the 174-kb and 142-kb 0ORI ${Delta}$ 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 0ORI ${Delta}$ and 5ORI ${Delta}$ 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.

DISCUSSION

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DISCUSSION

Yeast chromosomes are very stable, with loss rates in the rangeof 10^–5 loss per cell division. The loss rate of naturalyeast chromosomes is three orders of magnitude lower than theloss rates of centromeric plasmids or yeast artificial chromosomes(YACs) carrying foreign DNA (59, 60), providing an opportunityto measure very small perturbations in their stability. Oursystematic analysis of the effects of deleting replicators fromyeast chromosome III revealed that replicators are redundantin the sense that single ARS deletions and all combinationsof double, triple, and quadruple ARS deletions tested had nodetectable effect on chromosome stability. It was not untilwe deleted the five efficient replicators present in the leftmost71% of chromosome III that the loss rate of the chromosome increasedtwo- to threefold. This low loss rate raises the possibilitythat this 225-kb region could be replicated by a fork originatingat ARS315. Based on measured fork rates and length of S phase,the replication of this length of DNA by a single fork is feasible(43).

Further analysis of this 315-kb full-length 5ORI ${Delta}$ chromosomeand the 6ORI ${Delta}$ chromosome that also lacks ARS308 demonstratedthat two "dormant" replicators flanking HML, ARS301, and ARS303,which normally do not function as chromosomal replication origins(15), become weakly active and contribute to the maintenanceof the chromosome. These results are consistent with those ofVujcic et al. (63), who showed that these two ARS elements becomeactive in a chromosome from which the early-initiating replicatorsARS305 and ARS306 had been deleted. The region of chromosomeIII containing these dormant replicators is normally replicatedby a fork that originates at ARS305 and requires about 15 minto traverse the ca. 25-kb region between ARS305 and ARS303.ARS301 initiates replication late in a plasmid context (4),suggesting that it is likely to be replicated by a fork fromARS305 before it initiates replication in the chromosome. Supportfor this hypothesis is provided by studies on rad53 strains,in which replication forks were induced to collapse by treatmentwith hydroxyurea (31, 45). Under these conditions, which furtherslow forks initiated at ARS305, ARS301 initiated 30 to 45 minlater than ARS305. These observations suggest that replicatorsthat have evolved to initiate late may play an important rolein allowing complete replication of chromosomes in which early-initiatingreplication forks have collapsed. Because prereplicative complexescompetent to initiate replication are assembled only duringG₁ (reviewed in reference 2), regions near the ends of chromosomesthat are normally replicated by forks from interior originsmay be particularly vulnerable to replication fork collapse.In this regard it is interesting that Wyrick et al. (64) identifieda large number of pro-ARSs, defined as genomic sites that bindboth ORC and MCM proteins, in regions within 20 kb of telomeres.

As expected, we found that removing the right arm of the chromosomedistal to the ARS310 deletion, which contains efficient replicationorigins, increased the loss rates of the 5ORI ${Delta}$ and 6ORI ${Delta}$ fragmentsbut only to the extent caused by deleting the dormant originson the left end. An analysis of the direction of fork movementat several places along the 6ORI ${Delta}$ fragment provided evidencefor the activation of the dormant origins flanking HML, whichare likely to be the sources of forks moving rightward throughthe fragment. It also revealed forks moving in both directionsin the other three regions analyzed, including a region only15 kb from the telomere seeded by telomeric repeats in the fragmentationvector, suggesting that replication might initiate at or nearthe new telomere. Consistent with these results, in their analysisof a YAC in which they created a 174-kb region devoid of replicationorigins, van Brabant et al. (59) found forks moving in bothdirections through the originless region, including a regionnear the right telomere of the YAC. The mechanism by which suchinitiation events occur remains to be determined, although ourfindings provide some additional insight into the process.

Our analysis of the maintenance of a 61-kb linear chromosomefragment derived from the 61-kb ring chromosome that was formedby recombination between Ty elements to the left of LEU2 andto the right of PGK1 (52) provides evidence that either a lineartopology or the presence of functional telomeres allows replicationinitiation events that do not occur on the ring chromosome (Fig.5 and Table 2). One possible explanation for the stability ofthe 3ORI ${Delta}$ 61-kb linear chromosome fragment was that it was stabilizedby the addition of TRSs, which are added by telomerase duringthe formation of new telomeres. Longtine et al. (30) found thatthe addition of a TRS to a small ARS plasmid lacking a centromereincreased the stability of the plasmid by improving its segregationefficiency. However, when a TRS was included in a small plasmidcarrying a centromere, it caused a reduction in the stabilityof the plasmid. Consistent with the plasmid studies, we foundthat the insertion of a TRS in the 0ORI ${Delta}$ ring or in two 1ORI ${Delta}$ rings failed to improve the stability of the rings and may havecaused a small increase in their loss rates. Therefore, an internalTRS, which binds Rap1p and may recruit factors required fortelomeric silencing (29), is not sufficient to stabilize thering chromosome.

Further experiments will be required to determine the role oftelomeres and/or linear topology in the maintenance of the ORI ${Delta}$ chromosome fragments. One explanation of the role of telomeresis based on the observation that yeast telomeres acquire longerG-strand tails late during S phase, apparently as a result ofdegradation of the telomeric C-rich strand (reviewed in reference6). This observation indicates that the lagging strand replicationmachinery is active at most or all telomeres late in S phase,and it is possible that a replication fork can be formed attelomeres of chromosomes that have not replicated normally duringS phase. Another possibility is that ORC, which is known toplay a role in the establishment of transcriptionally represseddomains in yeast and Drosophila (reviewed in reference 1), isrecruited to telomeres by interaction with one or more telomerebinding proteins (see below).

Our finding that ORC is required for the maintenance of the141-kb 5ORI ${Delta}$ chromosome suggests that the normal replicationinitiation machinery is required for the maintenance of thefragment, although we cannot eliminate the possibility thatthe role ORC plays is distinct from that of replication initiation(see below). It is unlikely that additional ARS elements thatcontribute to the maintenance of the 141-kb 6ORI ${Delta}$ are present.In our analysis of overlapping fragments of chromosome III forARS activity (37, 41), we found no evidence of additional ARSelements, although recent genome-wide analyses of potentialreplication origins in budding yeast have identified additionalpotential replicators on chromosome III. In their analyses ofORC and MCM binding sites, Wyrick et al. (64) and Xu et al.(65)found a single ORC-MCM binding site present in the 142-kb chromosomeIII fragment that was not associated with an ARS element. Thissite is a few kilobases distal to ARS309, and a fragment amplifiedfrom this region did not have ARS activity in the plasmid assay(65). Moreover, initiation events at this site cannot accountfor the leftward-moving forks seen centromere proximal to theARS310 deletion (Fig. 4). Therefore, if replication is initiatingat internal sites in an ORC-dependent manner, the sites mustnot bind ORC efficiently enough to be detected in the chromatinimmunoprecipitation assay used to identify ORC binding sites.

In another approach to identifying replication origins, Nieduszynskiet al. (39) used an analysis of phylogenetic conservation ofintergenic regions to identify replication origin sequencesin S. cerevisiae. This analysis identified most of the knownARS elements on chromosome III but no additional ones. An additionalfive phylogenetically conserved intergenic regions with matchesto the ACS are present in the chromosome fragment studied here,but they have not been tested for ARS activity (C. A. Nieduszynski,personal communication). These sites were eliminated from thefinal list of replication origins by one or more of the filtersutilized in the study (39). Finally, the results of the whole-genomeapproaches to mapping replication origins discussed in the introductionhave recently been compiled with both the ORC and MCM bindingstudies and the phylogenetic conservation data in an origindatabase, OriDB (38). This database lists four potential replicationinitiation sites on chromosome III that were identified in oneor more microarray studies. Only one of these sites was identifiedas an ORC-MCM binding site, and the fragment containing thisregion does not have ARS activity.

In addition to its well-defined role in replication initiationand in the establishment of transcriptionally repressed domainsin yeast and Drosophila (reviewed in reference 1), ORC is alsobelieved to have a mitotic function. Dillin and Rine (11) firstsuggested a mitotic role for ORC based on the mitotic delayseen in the orc5-1mutant. The orc2-1 and orc5-1 mutations areeach synthetically lethal with deletion mutations causing sisterchromatid cohesion defects, including ctf4 ${Delta}$ , ctf8 ${Delta}$ , ctf18 ${Delta}$ , anddcc1 ${Delta}$ (53). In addition, the orc5-1 mutation enhances the cohesiondefect caused by scc1-73, a mutation in a cohesin subunit. Finally,ORC has also been shown to play a cohesin-independent role insister chromatid cohesion in yeast (46). We cannot eliminatethe possibility that the role of ORC in maintenance of the 142-kb5ORI ${Delta}$ fragment is related to its role in transcriptional silencingor sister chromatid cohesion. To ultimately understand wherereplication initiates on the 5ORI ${Delta}$ and 6ORI ${Delta}$ fragments will requirethe analysis of single molecules. We are currently using single-moleculeanalysis of the replicated DNA (40) to analyze the replicationof individual originless chromosome III fragments.

In their studies of a YAC carrying human DNA similar in compositionto yeast DNA, van Brabant et al. (59) found that an "originless"derivative of this YAC was lost at a rate similar to the lossrate of our 142-kb 6ORI ${Delta}$ chromosome III fragment lacking bothdormant replicators and replicators on the right arm. They foundthat maintenance of the YAC required RAD9, which encodes a mediatorprotein required for the DNA damage checkpoint (reviewed inreference 28). In a rad9 ${Delta}$ strain, the YAC was unstable when cellswere grown under selection for the YAC, with derivatives thathad deleted large portions of the originless region overtakingthe population, indicating that the DNA damage checkpoint isimportant for maintaining the YAC. Consistent with their results,we have found that RAD9 and other genes encoding componentsof the DNA damage checkpoint pathway are important for the maintenanceof the originless chromosome fragment (unpublished data). Thesefindings raise the possibility that DNA damage checkpoint pathwayhas a role in an alternative route to replication initiation.

ACKNOWLEDGMENTS

We thank K. Runge, V. Zakian, A. Dillin, and J. Rine for plasmids;Conrad Nieduszynski for additional information about his phylogeneticanalysis of conserved sequences on chromosome III; ShakespeareSajous for technical assistance; Michael Newlon, Carmela Irene,and James Theis for helpful comments on the manuscript; andcurrent and past members of the Newlon lab for helpful discussions.

This work was supported by a NIH grant (GM35679) awarded toC.S.N.

FOOTNOTES

* Corresponding author. Mailing address: Department of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, ICPH, 225 Warren St., Newark, NJ 07103. Phone: (973) 972-4483, ext. 24227. Fax: (973) 972-1286. E-mail: newlon{at}umdnj.edu

Published ahead of print on 23 April 2007.

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

Present address: Department of Physiology and Biophysics, WeillMedical College of Cornell University, 1300 York Ave., New York,NY 10021.

Present address: Department of Oncological Sciences, HuntsmanCancer Institute, University of Utah, 2000 Circle of Hope, SaltLake City, UT 84112.

^¶ Present address: SciMed Technologies Sdn. Bhd., 1F-08 IOI BusinessPark, No. 1 Persiaran Puchong Jaya Selatan, Bandar Puchong Jaya,47100 Puchong, Selangor, Malaysia.

REFERENCES

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