Roles for internal and flanking sequences in regulating the activity of mating-type-silencer-associated replication origins in Saccharomyces cerevisiae

ARS301 and ARS302 are inactive replication origins located at the left end of budding yeast (Saccharomyces cerevisiae) chromosome III, where they are associated with the HML-E and -I silencers of the HML mating type cassette. Although they function as replication origins in plasmids, they do not serve as origins in their normal chromosomal locations, because they are programmed to fire so late in S phase that they are passively replicated by the replication fork from neighboring early-firing ARS305 before they have a chance to fire on their own. We asked whether the nucleotide sequences required for plasmid origin function of these silencer-associated chromosomally inactive origins differ from the sequences needed for plasmid origin function by nonsilencer-associated chromosomally active origins. We could not detect consistent differences in sequence requirements for the two types of origins. Next, we asked whether sequences within or flanking these origins are responsible for their chromosomal inactivity. Our results demonstrate that both flanking and internal sequences contribute to chromosomal inactivity, presumably by programming these origins to fire late in S phase. In ARS301, the function of the internal sequences determining chromosomal inactivity is dependent on the checkpoint proteins Mec1p and Rad53p.     

Active Role of a Human Genomic Insert in Replication of a Yeast Artificial Chromosome

Yeast artificial chromosomes (YACs) are a common tool for cloning eukaryotic DNA. The manner by which large pieces of foreign DNA are assimilated by yeast cells into a functional chromosome is poorly understood, as is the reason why some of them are stably maintained and some are not. We examined the replication of a stable YAC containing a 240-kb insert of DNA from the human T-cell receptor beta locus. The human insert contains multiple sites that serve as origins of replication. The activity of these origins appears to require the yeast ARS consensus sequence and, as with yeast origins, additional flanking sequences. In addition, the origins in the human insert exhibit a spacing, a range of activation efficiencies, and a variation in times of activation during S phase similar to those found for normal yeast chromosomes. We propose that an appropriate combination of replication origin density, activation times, and initiation efficiencies is necessary for the successful maintenance of YAC inserts.     

Replicator dominance in a eukaryotic chromosome.

Replicators are genetic elements that control initiation at an origin of DNA replication (ori). They were first identified in the yeast Saccharomyces cerevisiae as autonomously replicating sequences (ARSs) that confer on a plasmid the ability to replicate in the S phase of the cell cycle. The DNA sequences required for ARS function on a plasmid have been defined, but because many sequences that participate in ARS activity are not components of chromosomal replicators, a mutational analysis of the ARS1 replicator located on chromosome IV of S. cerevisiae was performed. The results of this analysis indicate that four DNA elements (A, B1, B2 and B3) are either essential or important for ori activation in the chromosome. In a yeast strain containing two closely spaced and identical copies of the ARS1 replicator in the chromosome, only one is active. The mechanism of replicator repression requires the essential A element of the active replicator. This element is the binding site for the origin recognition complex (ORC), a putative initiator protein. The process that determines which replicator is used, however, depends entirely upon flanking DNA sequences.     

Time of replication of yeast centromeres and telomeres

The time of replication of centromeres and telomeres of the yeast S. cerevisiae was determined by performing Meselson-Stahl experiments with synchronized cells. The nine centromeres examined become hybrid in density early in S phase, eliminating the possibility that a delay in the replication of centromeres until mitosis is responsible for sister chromatid adherence and proper chromosome segregation at anaphase. The conserved sequence element Y', present at most telomeres, replicates late in S phase, as do the unique sequences adjacent to five specific telomeres. The early and late replication times of these structural elements may be either essential for their proper function or a consequence of some architectural feature of the chromosome.     

Time of replication of ARS elements along yeast chromosome III.

The replication of putative replication origins (ARS elements) was examined for 200 kilobases of chromosome III of Saccharomyces cerevisiae. By using synchronous cultures and transfers from dense to light isotope medium, the temporal pattern of mitotic DNA replication of eight fragments that contain ARSs was determined. ARS elements near the telomeres replicated late in S phase, while internal ARS elements replicated in the first half of S phase. The results suggest that some ARS elements in the chromosome may be inactive as replication origins. The actively expressed mating type locus, MAT, replicated early in S phase, while the silent cassettes, HML and HMR, replicated late. Unexpectedly, chromosome III sequences were found to replicate late in G1 at the arrest induced by the temperature-sensitive cdc7 allele.     

Replication in yeasts of plasmid pE194 from Staphylococcus aureus

The ability of the plasmid pE194 from S. aureus to serve as an autonomously replicating sequence (ARS) in yeast was shown. The hybrid plasmid pLD744 that contains pE194 and the yeast LEU2 gene sequences is unstable in yeast like other YRp-vectors: the mitotic stability of the pLD744 was as much as 1%. The plasmid pLD712 that differs from pLD744 by the existence of a centromeric sequence from the chromosome III of yeast Saccharomyces cerevisiae reveals about one order greater stability. The observation that there are some sequences in the primary structure of the pE194 which strongly conform to the ARS consensus in yeast inclines us to infer that the existence of ARS consensus on pE194 DNA is not sufficient for its effective replication in yeast.     

Yeast DNA replication in vitro: initiation and elongation events mimic in vivo processes

An enzyme system prepared from Saccharomyces cerevisiae carries out the replication of exogenous yeast plasmid DNA. Replication in vitro mimics that in vivo in that DNA synthesis in extracts of strain cdc8, a temperature-sensitive DNA replication mutant, is thermolabile relative to the wild-type, and in that aphidicolin inhibits replication in vitro. Furthermore, only plasmids containing a functional yeast replicator, ARS, initiate replication at a specific site in vitro. Analysis of replicative intermediates shows that plasmid YRp7, which contains the chromosomal replicator ARS1, initiates bidirectional replication in a 100 bp region within the sequence required for autonomous replication in vivo. Plasmids containing ARS2, another chromosomal replicator, and the ARS region of the endogenous yeast plasmid 2 microns circle give similar results, suggesting that ARS sequences are specific origins of chromosomal replication. Used in conjunction with deletion mapping, the in vitro system allows definition of the minimal sequences required for the initiation of replication.     

A position effect on the time of replication origin activation in yeast.

The chromosomes of eukaryotes are characterized by the mosaic nature of their replication--large regions of DNA that replicate early in S phase are interspersed with regions that replicate late. This pattern of early and late synthesis appears to be the consequence of a temporal program that activates replication origins at different times. The basis of this temporal regulation in the yeast S. cerevisiae has been investigated by changing the chromosomal locations of two origins, one activated early in the S phase (ARS1) and one activated late (ARS501). We show that the cis-acting information controlling time of activation can be separated from the element that determines origin function. For the ARS501 origin, late activation appears to be a consequence of its proximity to the telomere.     

Chromosomal DNA replication initiates at the same origins in meiosis and mitosis.

Autonomously replicating sequence (ARS) elements are identified by their ability to promote high-frequency transformation and extrachromosomal replication of plasmids in the yeast Saccharomyces cerevisiae. Six of the 14 ARS elements present in a 200-kb region of Saccharomyces cerevisiae chromosome III are mitotic chromosomal replication origins. The unexpected observation that eight ARS elements do not function at detectable levels as chromosomal replication origins during mitotic growth suggested that these ARS elements may function as chromosomal origins during premeiotic S phase. Two-dimensional agarose gel electrophoresis was used to map premeiotic replication origins in a 100-kb segment of chromosome III between HML and CEN3. The pattern of origin usage in premeiotic S phase was identical to that in mitotic S phase, with the possible exception of ARS308, which is an inefficient mitotic origin associated with CEN3. CEN3 was found to replicate during premeiotic S phase, demonstrating that the failure of sister chromatids to disjoin during the meiosis I division is not due to unreplicated centromeres. No origins were found in the DNA fragments without ARS function. Thus, in both mitosis and meiosis, chromosomal replication origins are coincident with ARS elements but not all ARS elements have chromosomal origin function. The efficiency of origin use and the patterns of replication termination are similar in meiosis and in mitosis. DNA replication termination occurs over a broad distance between active origins.     

Evidence for the involvement of a single major species of replicative complex in DNA synthesis from two diverse nuclear replicons in yeast

The activity that replicates the 2-micron yeast DNA plasmid in vitro can be isolated as a high-molecular weight (approximately 2 X 10(6)) fraction, which possesses many of the features of a multiprotein replicative complex. This fraction also initiates DNA synthesis at the yeast chromosomal replicator ARS1 raising the question whether the preparations discriminate between origins. It was determined that the binding of replicative complex to plasmids containing either 2-microns or ARS1 origins of replication was indistinguishable. The preparations also showed no preference among them for replication. In addition, the DNAs competed with each other to the same extent for binding of replicative complex. These results suggest that these two origins share one major species of replicative complex.     

Origin activation and formation of single-strand TG1-3 tails occur sequentially in late S phase on a yeast linear plasmid.

In order to understand the mechanisms leading to the complete duplication of linear eukaryotic chromosomes, the temporal order of the events involved in replication of a 7.5-kb Saccharomyces cerevisiae linear plasmid called YLpFAT10 was determined. Two-dimensional agarose gel electrophoresis was used to map the position of the replication origin and the direction of replication fork movement through the plasmid. Replication began near the center of YLpFAT10 at the site in the 2 microns sequences that corresponds to the 2 microns origin of DNA replication. Replication forks proceeded bidirectionally from the origin to the ends of YLpFAT10. Thus, yeast telomeres do not themselves act as origins of DNA replication. The time of origin utilization on YLpFAT10 and on circular 2 microns DNA in the same cells was determined both by two-dimensional gel electrophoresis and by density transfer experiments. As expected, 2 microns DNA replicated in early S phase. However, replication of YLpFAT10 occurred in late S phase. Thus, the time of activation of the 2 microns origin depended upon its physical context. Density transfer experiments established that the acquisition of telomeric TG1-3 single-strand tails, a predicted intermediate in telomere replication, occurred immediately after the replication forks approached the ends of YLpFAT10. Thus, telomere replication may be the very last step in S phase.     

Conservation of ARS elements and chromosomal DNA replication origins on chromosomes III of Saccharomyces cerevisiae and S. carlsbergensis.

DNA replication origins, specified by ARS elements in Saccharomyces cerevisiae, play an essential role in the stable transmission of chromosomes. Little is known about the evolution of ARS elements. We have isolated and characterized ARS elements from a chromosome III recovered from an alloploid Carlsberg brewing yeast that has diverged from its S. cerevisiae homeologue. The positions of seven ARS elements identified in this S. carlsbergensis chromosome are conserved: they are located in intergenic regions flanked by open reading frames homologous to those that flank seven ARS elements of the S. cerevisiae chromosome. The S. carlsbergensis ARS elements were active both in S. cerevisiae and S. monacensis, which has been proposed to be the source of the diverged genome present in brewing yeast. Moreover, their function as chromosomal replication origins correlated strongly with the activity of S. cerevisiae ARS elements, demonstrating the conservation of ARS activity and replication origin function in these two species.     

Identification of a predominant replication origin in fission yeast.

We have identified five autonomously replicating sequences (ARSs) in a 100 kbp region of the Schizosaccharomyces pombe chromosome II. Analyses of replicative intermediates of the chromosome DNA by neutral/neutral two-dimensional gel electrophoresis demonstrated that at least three of these ARS loci operate as chromosomal replication origins. One of the loci,ori2004, was utilized in almost every cell cycle, while the others were used less frequently. The frequency of initiation from the respective chromosomal replication origin was found to be roughly proportional to the efficiency of autonomous replication of the corresponding ARS plasmid. Replication from ori2004 was initiated within a distinct region almost the same as that for replication of the ARS plasmid. These results showed that the ori2004 region of approximately 3 kbp contains all the cis elements essential for initiation of chromosome replication.     

Multiple DNA elements in ARS305 determine replication origin activity in a yeast chromosome.

A yeast autonomously replicating sequence, ARS305, shares essential components with a chromosome III replicator, ORI305. Known components include an ARS consensus sequence (ACS) element, presumed to bind the origin recognition complex (ORC), and a broad 3'-flanking sequence which contains a DNA unwinding element. Here linker substitution mutagenesis of ARS305 and analysis of plasmid mitotic stability identified three short sequence elements within the broad 3'-flanking sequence. The major functional element resides directly 3' of the ACS and the two remaining elements reside further downstream, all within non-conserved ARS sequences. To determine the contribution of the elements to replication origin function in the chromosome, selected linker mutations were transplaced into the ORI305 locus and two-dimensional gel electrophoresis was used to analyze replication bubble formation and fork directions. Mutation of the major functional element identified in the plasmid mitotic stability assay inactivated replication origin function in the chromosome. Mutation of each of the two remaining elements diminished both plasmid ARS and chromosomal origin activities to similar levels. Thus multiple DNA elements identified in the plasmid ARS are determinants of replication origin function in the natural context of the chromosome. Comparison with two other genetically defined chromosomal replicators reveals a conservation of functional elements known to bind ORC, but no two replicators are identical in the arrangement of elements downstream of ORC binding elements or in the extent of functional sequences adjacent to the ACS.     

Replication of each copy of the yeast 2 micron DNA plasmid occurs during the S phase.

Saccharomyces cerevisiae contains 50-100 copies per cell of a circular plasmid called 2 micron DNA. Replication of this DNA was studied in two ways. The distribution of replication events among 2 micron DNA molecules was examined by density transfer experiments with asynchronous cultures. The data show that 2 micron DNA replication is similar to chromosomal DNA replication: essentially all 2 micron duplexes were of hybrid density at one cell doubling after the density transfer, with the majority having one fully dense strand and one fully light strand. The results show that replication of 2 micron DNA occurs by a semiconservative mechanism where each of the plasmid molecules replicates once each cell cycle. 2 micron DNA is the only known example of a multiple-copy, extrachromosomal DNA in which every molecule replicates in each cell cycle. Quantitative analysis of the data indicates that 2 micron DNA replication is limited to a fraction of the cell cycle. The period in the cell cycle when 2 micron DNA replicates was examined directly with synchronous cell cultures. Synchronization was accomplished by sequentially arresting cells in G1 phase using the yeast pheromone alpha-factor and incubating at the restrictive temperature for a cell cycle (cdc 7) mutant. Replication was monitored by adding 3H-uracil to cells previously labeled with 14C-uracil, and determining the 3H/14C ratio for purified DNA species. 2 micron DNA replication did not occur during the G1 arrest periods. However, the population of 2 micron DNA doubled during the synchronous S phase at the permissive temperature, with most of the replication occurring in the first third of S phase. Our results indicate that a mechanism exists which insures that the origin of replication of each 2 micron DNA molecule is activated each S phase. As with chromosomal DNA, further activation is prevented until the next cell cycle. We propose that the mechanism which controls the replication initiation of each 2 micron DNA molecule is identical to that which controls the initiation of chromosomal DNA.     

Comparison of the two major ARS elements of the ura4 replication origin region with other ARS elements in the fission yeast,Schizosaccharomyces pombe

We have previously peported that the replication orgin region located near the ura4 gene on chromosome III of the fission yeast, Schizosaccharomyces pombe, contains three closely spaced origins, each associated with an autonomously replicating sequence (ARS) element. Here we report the nucleotide sequences of two of these ARS elements, ars3002 and ars3003. The two ARS elements are located on either side of a transcribed 1.5 kb open reading frame. Like 11 other S. pombe ARS elements whose sequences have previously been determined in other laboratories, the 2 new ARS elements are unusually A+T-rich. All 13 ARS elements contain easily unwound stretches of DNA. Each of the ARS elements contains numerous copies, at a higher than expected frequency, of short stretches of A+T-rich DNA in which most of the Ts are on one strand and most of the As are on the complementary strand. We discuss the potential significance for ARS function of these multiple asymmetric A+T-rich sequences.     

Cloning of Lentinus edodes mitochondrial DNA fragment capable of autonomous replication in Saccharomyces cerevisiae

Mitochondrial (mt) DNA of the higher basidiomycetes Lentinus edodes with a molecular weight of about 69 kb was partially digested with Sau3AI, cloned with plasmid YIp32 (a hybrid of pBR322 and the yeast leu2 gene) and analyzed for sequences capable of autonomous replication (ARSs) in the eukaryote Saccharomyces cerevisiae. One recombinant plasmid was isolated which contained 3.2 kb fragment of the mtDNA with ARS activity. This plasmid (named pSK52) exhibited a high-frequency yeast transformation and was found to be maintained within the cell as an extrachromosomal element. The stability and copy number properties of pSK52 were similar to those of the recombinant plasmid of YIp32 and S. cerevisiae mt-ARS constructed as a reference. Subcloning experiments were carried out to assess the localization of ARS on the above 3.2 kb fragment, revealing that the fragment contains at least two ARSs.     

Three ARS elements contribute to the ura4 replication origin region in the fission yeast, Schizosaccharomyces pombe.

The ura4 replication origin region, which is located near the ura4 gene on chromosome III of the fission yeast, Schizosaccharomyces pombe, contains multiple initiation sites. We have used 2D gel electrophoretic replicon mapping methods to study the distribution of these initiation sites, and have found that they are concentrated near three ARS elements (stretches of DNA which permit autonomous plasmid replication). To determine the roles of these ARS elements in the function of the ura4 origin region, we deleted either one or two of them from the chromosome and then assessed the consequences of the deletions by 2D gel electrophoresis. The results suggest that each of the three ARS elements is responsible for the initiation events in its vicinity and that the ARS elements interfere with each other in a hierarchical fashion. It is possible that the large initiation zones of animal cells are similarly composed of multiple mutually interfering origins.