Single-stranded-DNA-binding protein-dependent DNA unwinding of the yeast ARS1 region.

DNA unwinding of autonomously replicating sequence 1 (ARS1) from the yeast Saccharomyces cerevisiae was investigated. When a negatively supercoiled plasmid DNA containing ARS1 was digested with single-strand-specific mung bean nuclease, a discrete region in the vector DNA was preferentially digested. The regions containing the core consensus A domain and the 3'-flanking B domain of ARS1 were weakly digested. When the DNA was incubated with the multisubunit single-stranded DNA-binding protein (SSB, also called RPA [replication protein A]) from human and yeast cells prior to mung bean nuclease digestion, the cleavage in the A and B domains was greatly increased. Furthermore, a region corresponding to the 5'-flanking C domain of ARS1 was digested. These results indicate that three domains of ARS1, each of which is important for replication in yeast cells, closely correspond to the regions where the DNA duplex is easily unwound by torsional stress. SSB may stimulate the unwinding of the ARS1 region by its preferential binding to the destabilized three domains. Mung bean nuclease digestion of the substitution mutants with mutations of ARS1 (Y. Marahrens and B. Stillman, Science 255:817-823, 1992) revealed that the sequences in the B2 and A elements are responsible for the unwinding of the B domain and the region containing the A domain, respectively.     

The DNA unwinding element in a yeast replication origin functions independently of easily unwound sequences present elsewhere on a plasmid.

We have previously identified a DNA unwinding element (DUE) in autonomously replicating sequences (ARSs) and demonstrated a correlation between single-strand-specific nuclease hypersensitivity of the DUE and ARS-mediated plasmid replication in yeast. The DUE in the H4 ARS is the most easily unwound sequence in a supercoiled DNA molecule, in the context of the Ylp5 plasmid. To determine whether sequences which are more readily unwound than the ARS can influence replication activity, we have inserted such sequences, called 'torsional sinks', into the plasmids at a site distal to the ARS. We show that the torsional sink sequences effect reduction or elimination of the nuclease hypersensitivity of a variety of H4 ARS derivatives. However, we detect no difference in the in vivo replication activity of an individual ARS plasmid with or without a torsional sink. Thus, the function of the DUE in a yeast replication origin is unaffected by easily unwound sequences present elsewhere on the same plasmid.     

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.     

Ease of DNA unwinding is a conserved property of yeast replication origins.

Autonomously replicating sequence (ARS) elements function as plasmid replication origins. Our studies of the H4 ARS and ARS307 have established the requirement for a DNA unwinding element (DUE), a broad easily-unwound sequence 3' to the essential consensus that likely facilitates opening of the origin. In this report, we examine the intrinsic ease of unwinding a variety of ARS elements using (1) a single-strand-specific nuclease to probe for DNA unwinding in a negatively-supercoiled plasmid, and (2) a computer program that calculates DNA helical stability from the nucleotide sequence. ARS elements that are associated with replication origins on chromosome III are nuclease hypersensitive, and the helical stability minima correctly predict the location and hierarchy of the hypersensitive sites. All well-studied ARS elements in which the essential consensus sequence has been identified by mutational analysis contain a 100-bp region of low helical stability immediately 3' to the consensus, as do ARS elements created by mutation within the prokaryotic M13 vector. The level of helical stability is, in all cases, below that of ARS307 derivatives inactivated by mutations in the DUE. Our findings indicate that the ease of DNA unwinding at the broad region directly 3' to the ARS consensus is a conserved property of yeast replication origins.     

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.     

ARS replication during the yeast S phase

A 1.45 kb circular plasmid derived from yeast chromosome IV contains the autonomous replication element called ARS1. Isotope density transfer experiments show that each plasmid molecule replicates once each S phase, with initiation depending on two genetically defined steps required for nuclear DNA replication. A density transfer experiment with synchronized cells demonstrates that the ARS1 plasmid population replicates early in the S phase. The sequences adjacent to ARS1 on chromosome IV also initiate replication early, suggesting that the ARS1 plasmid contains information which determines its time of replication. The times of replication for two other yeast chromosome sequences, ARS2 and a sequence referred to as 1OZ, indicate that the temporal order of replication is ARS1 leads to ARS2 leads to 1OZ. These experiments show directly that specific chromosome regions replicate at specific times during the yeast S phase. If ARS elements are origins of chromosome replication, then the experiment reveals times of activation for two origins.     

A linear shuttle vector for yeast and the hypotrichous ciliate Stylonychia

A linear plasmid was constructed in vitro using the telomeres of the rDNA of Tetrahymena pyriformis. These telomeres were added to a yeast circular vector containing an ARS sequence from Dictyostelium, the LEU2 gene of yeast and the neo gene from Escherichia coli Tn5 fused with a eukaryotic promoter. The resulting plasmid was used to transform yeast. During the replication of the linear plasmid in yeast it was spontaneously modified at the extremity by the addition of 300 bp of yeast telomeric sequence for each end. Total DNA prepared from yeast transformants was used to transform the hypotrichous ciliate Stylonychia lemnae. The same plasmid isolated from Stylonychia can again be replicated in yeast.     

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.     

The function of the nuclear matrix attachment region of silkworm rDNA as an autonomously replicating sequence in plasmid and chromosomal replication origin in yeast

Nuclear matrix attachment regions (MARs) play a crucial role in chromatin architecture, gene expression, and DNA replication. Although it is well known that yeast autonomously replicating sequences (ARSs) bind nuclear matrix and MARs also function as ARS elements in yeast, whether a heterologous MAR or ARS element acts as a replication origin in the chromosome has not been elucidated. We previously identified a MAR (rMAR) located in the nontranscribed spacer (NTS) of silkworm Attacus ricini rDNA. We report here that this rMAR contains 10 copies of ARS consensus sequence (ACS) and several DNA unwinding regions. The rMAR employs ARS activity in yeast and a rARS element locates in the 3(') region of the rMAR. Furthermore, we have also revealed that either the rMAR or the rARS element functions as a replication origin in the chromosome. Our results provide the first direct evidence to demonstrate that heterologous rMAR and rARS display chromosomal origin activity, suggesting that the chromosome structure and replication origin of rDNA reserve some common features during evolution.     

Drosophila ARSs contain the yeast ARS consensus sequence and a replication enhancer.

A number of restriction fragments that function as autonomously replicating sequences (ARSs) in yeast have been isolated from Drosophila melanogaster DNA. The behaviour in yeast of plasmids containing Drosophila ARS elements was studied and compared to that exhibited by the archetypal yeast ARS-1 plasmid. ARS functions were localised by subcloning and BAL-31 deletion analysis. These studies demonstrated the structural and functional complexity of Drosophila ARSs. Each Drosophila ARS element has at least two domains, one essential for replication (the replication sequence, RS) and a second (the replication enhancer, RE) which is essential for maximum function of the RS. The RS of three Drosophila ARSs was shown to contain a sequence identical to an 11 bp yeast ARS consensus sequence (5' A/T TTTATPuTTT A/T 3'). These observations lend support to the hypothesis that heterologous ARS elements may be of biological significance.     

The basidiomycete Lentinus edodes linear mitochondrial DNA plasmid contains a segment exhibiting a high autonomously replicating sequence activity in Saccharomyces cerevisiae.

A linear DNA plasmid, designated pLLE1, has been isolated from a mitochondrial fraction of a strain of Lentinus edodes. pLLE1(11.0 kbp) was sensitive to the 3'----5'-acting exonuclease III and resistant to the 5'----3'-acting lambda exonuclease. It showed no homology with mitochondrial and nuclear genomic DNAs of plasmidless strain as well as the pLLE1-harboring host strain of L. edodes. The 1434-bp fragment (sequences) capable of autonomous replication in the yeast Saccharomyces cerevisiae (ARSs) was cloned from pLLE1 DNA with YIp32 (pBR322 containing yeast LEU2 DNA), which displayed a high ARS activity. The cloned 1434-bp fragment was shown to lie near to the end of pLLE1 DNA (nucleotides about 800-2200) and contained three consecutive ARS consensus sequences (A/T)TTTAT(A/G)TTT(A/T) of S. cerevisiae and dispersive eight ARS consensus-like sequences. The subcloned 366-bp fragment containing the three ARSs retained original ARS activity of the 1434-bp fragment.     

Fine-structure analysis of the DNA sequence requirements for autonomous replication of Saccharomyces cerevisiae plasmids.

An autonomously replicating segment, ARS, is located 293 base pairs downstream from the histone H4 gene at the copy-I H3-H4 locus. The sequences needed for autonomous replication were defined by deletion analysis to include an ARS consensus sequence and an additional 3'-flanking region. External deletions into the 3'-flanking yeast sequences resulted in a loss of replication function. However, disruptions of the required 3'-flanking domain by either 10-base-pair linker-scanning substitutions or larger internal deletions did not impair autonomous replication. Thus, replication is dependent upon a flanking chromosome domain, but not an exact DNA sequence. The extent of the yeast sequences required in the 3'-flanking domain is variable depending on the nature of neighboring plasmid vector sequences. That is, there are certain vector sequences that prohibit replication when they are placed too close to the ARS consensus. These results suggest that the functional 3'-flanking domain of the H4 ARS is a specific DNA or chromatin structure or both.     

A reorganized Candida albicans DNA sequence promoting homologous non-integrative genetic transformation

In order to develop plasmids adequate for non-integrative genetic transformation of Candida albicans, a DNA fragment of 15.3 kb was cloned from this organism on the basis of its capacity to convert the integrative Saccharomyces cerevisiae vector YIp5 into a non-integrative one. Southern hybridization analysis, carried out with a labelled DNA probe of 3.6 kb derived from the cloned fragment, showed that it consisted of C. albicans DNA, the hybridization pattern indicating that the corresponding sequences were homologous to several chromosomal regions. The size of the C. albicans DNA promoting autonomous replication in S. cerevisiae was substantially reduced by subcloning. A 5.1 kb subfragment, defined by BamHI and SalI restriction sites, retained autonomous replication sequences (ARS) functional in the heterologous S. cerevisiae system and in C. albicans, when inserted in plasmid constructions that carried a S. cerevisiae trichodermin-resistance gene (tcm1) as selection marker. C. albicans transformants were both of the integrative and the non-integrative type and the plasmids recovered from the latter very often carried a reorganized ARS, indicating that recombination of the inserted ARS DNA had occurred in the homologous host. Successive reorganizations of the ARS insert in C. albicans eventually led to a more stable and much smaller fragment of 687 bp that was subsequently recovered unchanged from transformants. Sequence analysis of the 687 bp fragment revealed four 11-base blocks, rich in A+T, that carried the essential consensus sequence considered relevant for yeast ARS elements in addition to other features also described as characteristic of yeast replication origins.     

The replication behavior of Saccharomyces cerevisiae DNA in human cells

We studied the replication of random genomic DNA fragments from Saccharomyces cerevisiae in a long-term assay in human cells. Plasmids carrying large yeast DNA fragments were able to replicate autonomously in human cells. Efficiency of replication of yeast DNA fragments was comparable to that of similarly sized human DNA fragments and better than that of bacterial DNA. This result suggests that yeast genomic DNA contains sequence information needed for replication in human cells. To examine whether DNA replication in human cells would initiate specifically at a yeast origin of replication, we monitored initiation on a plasmid containing the yeast 2-micron autonomously replicating sequence (ARS) in yeast and human cells. We found that while replication initiates at the 2-micron ARS in yeast, it does not preferentially initiate at the ARS in human cells. This result suggests that the sequences that direct site specific replication initiation in yeast do not function in the same way in human cells, which initiate replication at a broader range of sequences.by J.A. Huberman     

The splicing factor SF2/ASF binds to ARS homologs in a human rDNA replication origin

The function of the relatively well-studied DNA replication origins in the yeast Saccharomyces cerevisiae is dependent upon interactions between origin replication complex (ORC) proteins and several defined origin sequence elements, including the 11 bp ARS consensus sequence (ACS). Although the ORC proteins, as well as numerous other protein components required for DNA replication initiation, are largely conserved between yeast and mammals, DNA sequences within mammalian replication origins are highly variable and sequences homologous to the yeast ACS elements are generally not present. We have previously identified several replication initiation sites within the nontranscribed spacer region of the human ribosomal RNA gene, and found that two highly utilized sites each contain a homologue of the yeast ACS embedded within a DNA unwinding element and a matrix attachment region. Here we examine protein binding within these initiation sites, and demonstrate that these ACS homologues specifically bind the alternate splicing factor SF2/ASF as well as GAPDH in vitro, and present evidence that the SF2/ASF interaction also occurs within the nuclei of intact cells. As the moderate upregulation of SF2/ASF has been linked to oncogenesis through the promotion of alternatively spliced forms of several regulatory proteins, our results suggest an additional mechanism by which SF2/ASF may influence the transformed cell phenotype.