Deletion mutations affecting autonomously replicating sequence ARS1 of Saccharomyces cerevisiae.

DNAs that contain specific yeast chromosomal sequences called ARSs transform Saccharomyces cerevisiae at high frequency and can replicate extrachromosomally as plasmids when introduced into S. cerevisiae by transformation. To determine the boundaries of the minimal sequences required for autonomous replication in S. cerevisiae, we have carried out in vitro mutagenesis of the first chromosomal ARS described, ARS1. Rather than identifying a distinct and continuous segment that mediates the ARS+ phenotype, we find three different functional domains within ARS1. We define domain A as the 11-base-pair (bp) sequence that is also found at most other ARS regions. It is necessary but not sufficient for high-frequency transformation. Domain B, which cannot mediate high-frequency transformation, or replicate by itself, is required for efficient, stable replication of plasmids containing domain A. Domain B, as we define it, is continuous with domain A in ARS1, but insertions of 4 bp between the two do not affect replication. The extent of domain B has an upper limit of 109 bp and a lower limit of 46 bp in size. There is no obvious sequence homology between domain B of ARS1 and any other ARS sequence. Finally, domain C is defined on the basis of our deletions as at least 200 bp flanking domain A on the opposite side from domain B and is also required for the stability of domain A in S. cerevisiae. The effect of deletions of domain C can be observed only in the absence of domain B, at least by the assays used in the current study, and the significance of this finding is discussed.     

Purification and characterization of OBF1: a Saccharomyces cerevisiae protein that binds to autonomously replicating sequences.

We previously identified a protein activity from Saccharomyces cerevisiae, OBF1, that bound specifically to a DNA element present in autonomously replicating sequences ARS120 and ARS121 (S. Eisenberg C. Civalier, and B. K. Tye, Proc. Natl. Acad. Sci. USA 85:743-746, 1988). OBF1 has now been purified to near homogeneity by conventional protein and DNA affinity chromatography. Electrophoresis of the purified protein in sodium dodecyl sulfate-polyacrylamide gels revealed the presence of two polypeptides. The major protein band had a relative molecular size of 123 kilodaltons, and the minor protein band, which constituted only a small fraction of total protein, had a molecular size of 127 kilodaltons. Both polypeptides cochromatographed with the specific ARS120 DNA-binding activity and formed a stable protein-DNA complex, isolatable by sedimentation through sucrose gradients. Using antibodies, we have shown that both polypeptides are associated with the isolated protein-DNA complexes. The ARS DNA-binding activity had a Stokes radius of 54 A (5.4 nm) and a sedimentation coefficient of 4.28S, as determined by gel filtration and sedimentation through glycerol gradients, respectively. These physical parameters, together with the denatured molecular size values, suggested that the proteins exist in solution as asymmetric monomers. Since both polypeptides recognized identical sequences and had similar physical properties, they are probably related. In addition to binding to ARS120, we found that purified OBF1 bounds with equal affinity to ARS121 and with 5- and 10-fold-lower affinity to ARS1 and HMRE, respectively. Furthermore, in the accompanying paper (S. S. Walker, S. C. Francesconi, B. K. Tye, and S. Eisenberg, Mol. Cell. Biol. 9:2914-2921, 1989), we demonstrate the existence of a high, direct correlation between the ability of purify OBF1 to bind to ARS121 and optimal in vivo ARS121 activity as an origin of replication. These findings, taken together, suggest a role for OBF1 in ARS function, presumably at the level of initiation of DNA replication at the ARS.     

Location and characterization of autonomously replicating sequences from chromosome VI of Saccharomyces cerevisiae.

We have reported the isolation of linking clones of HindIII and EcoRI fragments, altogether spanning a 230-kb continuous stretch of chromosome VI. The presence or absence of autonomously replicating sequence (ARS) activities in all of these fragments has been determined by using ARS searching vectors containing CEN4. Nine ARS fragments were identified, and their positions were mapped on the chromosome. Structures essential for and/or stimulative to ARS activity were determined for the ARS fragments by deletions and mutations. The organization of functional elements composed of core and stimulative sequences was found to be variable. Single core sequences were identified in eight of nine ARSs. The remaining ARS (ARS603) essential element is composed of two core-like sequences. The lengths of 3'- and 5'-flanking stimulative sequences required for the full activity of ARSs varied from ARS to ARS. Five ARSs required more than 100 bp of the 3'-flanking sequence as stimulative sequences, while not more than 79 bp of the 3' sequence was required by the other three ARSs. In addition, five ARSs had stimulative sequences varying from 127 to 312 bp in the 5'-flanking region of the core sequence. In general, these stimulative activities were correlated with low local delta Gs of unwinding, suggesting that the low local delta G of an ARS is an important element for determining the efficiency of initiation of replication of ARS plasmids.     

Saccharomyces cerevisiae RAP1 binds to telomeric sequences with spatial flexibility

A wide divergence has been detected in the telomeric sequences among budding yeast species. Despite their length and homogeneity differences, all these yeast telomeric sequences show a conserved core which closely matches the consensus RAP1-binding sequence. We demonstrate that the RAP1 protein binds this sequence core, without involving the diverged sequences outside the core. In Saccharomyces castellii and Saccharomyces dairensis specific classes of interspersed variant repeats are present. We show here that a RAP1-binding site is formed in these species by connecting two consecutive 8 bp telomeric repeats. DNase I footprint analyses specify the binding site as the 13 bp sequence CTGGGTGTCTGGG. The RAP1 protein also binds the variant repeats, although with a lowered affinity. However, a split footprint is produced when RAP1 binds a variant repeat where the two half-sites of the binding site are separated by an additional 6 nt. This is probably caused by the intervening sequence looping out of the RAP1-DNA complex. We suggest that the bipartite subdomain structure of the RAP1 protein allows it to remodel telomeric chromatin, a feature which may be of great relevance for telomeric chromatin assembly and structure in vivo.     

In addition to the ARS core, the ARS box is necessary for autonomously replicating sequences

Summary Autonomously replicating sequences (ARSs) were cloned from nuclear and mitochondrial DNA of D. melanogaster using YIp5, which is composed of pBR322 and the yeast ura3 gene, as the cloning vector and YNN27, a Ura^- yeast strain as the recipient. The nucleotide sequences of six ARSs, two from nuclear bulk, two from the nuclear 1.688 satellite, and two from mitochondorial DNA, were determined. The relationship between the transformation frequency and the inclusion of the ARS core, 5 _T ^A TT-TAT _A ^G TTT _T ^A 3, of these fragments was analysed. All the ARSs contained an ARS core or a single base change of it. However, not all the fragments that contained a single base change of the ARS core were able to transform the recipient cells, suggesting that certain bases in the ARS core were not exchangeable. It is suggested by transformation experiments with subfragments that in addition to an ARS core, an ARS box which is located within 25 bp upstream of the ARS core and whose sequence is composed of 5TNT _G ^A AA 3, is necessary for autonomous replication.     

Micronuclear DNA of Oxytricha nova contains sequences with autonomously replicating activity in Saccharomyces cerevisiae.

Oxytricha nova is a hypotrichous ciliate with micronuclei and macronuclei. Micronuclei, which contain large, chromosomal-sized DNA, are genetically inert but undergo meiosis and exchange during cell mating. Macronuclei, which contain only small, gene-sized DNA molecules, provide all of the nuclear RNA needed to run the cell. After cell mating the macronucleus is derived from a micronucleus, a derivation that includes excision of the genes from chromosomes and elimination of the remaining DNA. The eliminated DNA includes all of the repetitious sequences and approximately 95% of the unique sequences. We cloned large restriction fragments from the micronucleus that confer replication ability on a replication-deficient plasmid in Saccharomyces cerevisiae. Sequences that confer replication ability are called autonomously replicating sequences. The frequency and effectiveness of autonomously replicating sequences in micronuclear DNA are similar to those reported for DNAs of other organisms introduced into yeast cells. Of the 12 micronuclear fragments with autonomously replicating sequence activity, 9 also showed homology to macronuclear DNA, indicating that they contain a macronuclear gene sequence. We conclude from this that autonomously replicating sequence activity is nonrandomly distributed throughout micronuclear DNA and is preferentially associated with those regions of micronuclear DNA that contain genes.     

The chromatin structure of Saccharomyces cerevisiae autonomously replicating sequences changes during the cell division cycle.

The chromatin structures of two well-characterized autonomously replicating sequence (ARS) elements were examined at their chromosomal sites during the cell division cycle in Saccharomyces cerevisiae. The H4 ARS is located near one of the duplicate nonallelic histone H4 genes, while ARS1 is present near the TRP1 gene. Cells blocked in G1 either by alpha-factor arrest or by nitrogen starvation had two DNase I-hypersensitive sites of about equal intensity in the ARS element. This pattern of DNase I-hypersensitive sites was altered in synchronous cultures allowed to proceed into S phase. In addition to a general increase in DNase I sensitivity around the core consensus sequence, the DNase I-hypersensitive site closest to the core consensus became more nuclease sensitive than the distal site. This change in chromatin structure was restricted to the ARS region and depended on replication since cdc7 cells blocked near the time of replication initiation did not undergo the transition. Subsequent release of arrested cdc7 cells restored entry into S phase and was accompanied by the characteristic change in ARS chromatin structure.     

Difference in strength of autonomously replicating sequences among repeats in the rDNA region of Saccharomyces cerevisiae

The rDNA region of Saccharomyces cerevisiae contains 100-200 tandemly repeated copies of a 9 kb unit, each with a potential replication origin. In the present studies of cloned fragments from the region involved in the regulation of replication of rDNA, we detected differences in autonomously replicating sequence (ARS) activity for clones from the same yeast strain. One clone, which showed very low ARS activity, carried a point mutation, a C instead of T, in position 9 of the essential 11 bp consensus ARS as compared to clones carrying the normal 10-of-11-bp match to the consensus. The mutation could be traced back to genomic rDNA where it represents about one-third of the rDNA units in that strain. Differences in ARS activity have implications for understanding the regulation of replication of rDNA, and the ratio of active to inactive ARS in the rDNA region may be important for potential generation of extrachromosomal copies.     

The mitochondrial genome of Saccharomyces cerevisiae contains numerous, densely spaced autonomously replicating sequences

Restriction fragments produced by a complete Sau3A cleavage of Saccharomyces cerevisiae grande mitochondrial DNA were ligated into the yeast-Escherichia coli shuttle vector YIp5 to establish a clone library representing the mitochondrial genome. 30 hybrid plasmids with an average insert size of 1200 bp were chosen at random and tested for the presence of an autonomously replicating sequence (ars). Over two-thirds of these plasmids transformed yeast at high frequency, indicating the mitochondrial genome contains a large number of ars elements. Our calculations suggest there may be over 40 ars elements contained within the mitochondrial DNA with an average spacing of less than 1700 bp. Mapping experiments indicate that ars elements can be found at many locations on the mitochondrial genome, and in the initial example we have tested, the locations of ars elements derived from grande and petite mtDNAs appear to coincide. If we assume that these ars elements represent mitochondrial DNA replication origins used in vivo, these observations would explain in part the fact that petite mtDNAs can be derived from any location on the grande mitochondrial genome.     

Identification of autonomously replicating circular subtelomeric Y'' elements in Saccharomyces cerevisiae.

We marked a large number of yeast telomeres within their Y' regions by transforming strains with a fragment of Y' DNA into which the URA3 gene had been inserted. A few of the Ura+ transformants obtained were very unstable and were found to contain autonomously replicating URA3-marked circular Y' elements in high copy number. These marked extrachromosomal circles were capable of reintegrating into the chromosome at other telomeric locations. In contrast, most of the Ura+ transformants obtained were quite stable mitotically and were marked at bona fide chromosomal ends. These stable transformants gave rise to mitotically unstable URA3-marked circular Y' elements at a low frequency (up to 2.5%). The likelihood that such excisions and integrations represent a natural process in Saccharomyces cerevisiae is supported by our identification of putative Y' circles in untransformed strains. The transfer of Y' information among telomeres via a circular intermediate may be important for homogenizing the sequences at the ends of yeast chromosomes and for generating the frequent telomeric rearrangements that have been observed in S. cerevisiae.     

Isolation and nucleotide sequence of an autonomously replicating sequence (ARS) element functional in Candida albicans and Saccharomyces cerevisiae

Summary An 8.6-kb fragment was isolated from an EcoRI digest of Candida albicans ATCC 10261 genomic DNA which conferred the property of autonomous replication in Saccharomyces cervisiae on the otherwise non-replicative plasmid pMK155 (5.6 kb). The DNA responsible for the replicative function was subcloned as a 1.2-kb fragment onto a non-replicative plasmid (pRC3915) containing the C. albicans URA3 and LEU2 genes to form plasmid pRC3920. This plasmid was capable of autonomous replication in both S. cerevisiae and C. albicans and transformed S. cerevisiae AH22 (leu2 ^–) to Leu⁺ at a frequency of 2.15 × 10³ transformants per pg DNA, and transformed C. albicans SGY-243 (ura3) to Ura⁺ at a frequency of 1.91 × 10³ transformants per g DNA. Sequence analysis of the cloned DNA revealed the presence of two identical regions of eleven base pairs (5TTTTATGTTTT3) which agreed with the consensus of autonomously replicating sequence (ARS) cores functional in S. cerevisiae. In addition there were two 10/11 and numerous 9/11 matches to the core consensus. The two 11/11 matches to the consensus, CaARS1 and CaARS2, were located on opposite strands in a non-coding AT-rich region and were separated by 107 bp. Also present on the C. albicans DNA, 538 by from the ARS cores, was a gene for 5S rRNA which showed sequence homology with several other yeast 5S rRNA genes. A sub-fragment (494 bp) containing the 5S rRNA gene (but not the region containing the ARS cores) hybridized to genomic DNAs from a number of yeast species, including S. cerevisiae, C. tropicalis, C. pseudotropicalis, C. parapsilosis, C. kruseii, C. (Torulopsis) glabrata and Neurospora crassa. The 709-bp ARS element (but not the 5S rRNA gene) was necessary for high-frequency transformation and autonomous plasmid replication in both S. cerevisiae and C. albicans.EMBL/GenBank database accession number: X16634 (5S rRNA)     

A family of Saccharomyces cerevisiae repetitive autonomously replicating sequences that have very similar genomic environments

We have characterized a family of moderately repetitive autonomously replicating sequences (ARSs) in Saccharomyces cerevisiae. Restriction mapping, deletion studies and hybridization studies suggest that these ARSs, which are probably less than 350 base-pairs in size, share one common feature: each is located close to, but not within, a repetitive sequence (131) of approximately 10(3) to approximately 1.5 X 10(3) base-pairs in length. These ARSs can be divided into two classes (X and Y) by their sequence homology and genomic environments. Each of the class X ARSs is embedded within a repetitive sequence (X) of variable length (approximately 0.3 X 10(3) to approximately 3.75 X 10(3) base-pairs); each of the class Y ARSs is embedded within a highly conserved repetitive sequence (Y) of approximately 5.2 X 10(3) base-pairs in length. Both of these sequences are located directly adjacent to the 131 sequence.     

The Mcm467 complex of Saccharomyces cerevisiae is preferentially activated by autonomously replicating DNA sequences

We have analyzed the role of single-stranded DNA (ssDNA) in the modulation of the ATPase activity of Mcm467 helicase of the yeast Saccharomyces cerevisiae. The ATPase activity of the Mcm467 complex is modulated in a sequence-specific manner and that the ssDNA sequences derived from the origin of DNA replication of S. cerevisiae autonomously replicating sequence 1 (ARS1) are the most effective stimulators. Synthetic oligonucleotides, such as oligo(dA) and oligo(dT), also stimulated the ATPase activity of the Mcm467 complex, where oligo(dT) was more effective than oligo(dA). However, the preference of a thymidine stretch appeared unimportant, because with yeast ARS1 derived sequences, the A-rich strand was as effective in stimulating the ATPase activity, as was the T-rich strand. Both of these strands were more effective stimulators than either oligo(dA)( )()or oligo(dT). The DNA helicase activity of Mcm467 complex is also significantly stimulated by the ARS1-derived sequences. These results indicate that the ssDNA sequences containing A and B1 motifs of ARS1, activate the Mcm467 complex and stimulate its ATPase and DNA helicase activities. Our results also indicate that the yeast replication protein A stimulated the ATPase activity of the Mcm467 complex.     

A mutant that affects the function of autonomously replicating sequences in yeast

We previously reported the isolation of a series of mcm mutants that are defective in the maintenance of minichromosomes in yeast. These minichromosomes are circular plasmids, each containing an autonomously replicating sequence (ARS) and a centromere. One of the mcm mutants, mcm2, has the following phenotype: at room temperature it affects the stability of only some minichromosomes depending on the ARS present, while at high temperature it affects all minichromosomes tested irrespective of the ARS present. Here we show that the mcm defect as well as its temperature-dependent specificity for ARSs can be demonstrated with circular as well as linear plasmids that do not contain centromeric sequences. Larger chromosomes containing multiple ARSs are also unstable in this mutant. Further analyses indicate that the mcm2 mutation causes the loss, rather than the aberrant segregation, of the circular minichromosomes. In addition, this mutation appears to stimulate mitotic recombination frequencies. These properties of the mcm2 mutant are consistent with the idea that the mcm2 mutation results in a defect in the initiation of DNA replication at ARSs, the putative chromosomal replication origins in yeast.     

An autonomously replicating sequence from HeLa DNA shows a similar organization to the yeast ARS1 element

Summary A HeLa DNA fragment, which may function as an anchorage point to the nuclear matrix for human chromosomes 1 and 2, also functions as an autonomously replicating sequence (ARS) in the yeast Saccharomyces cerevisiae. In the present report we show that this DNA fragment contains both bent DNA and an A-T rich region which appear to be associated with the ARS function. More interestingly, DNA sequence analysis shows that the spatial distribution of these features is strikingly similar to that found in the yeast ARS1 element.