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.     

The inefficient replication origin from yeast ribosomal DNA is naturally impaired in the ARS consensus sequence and in DNA unwinding.

Ribosomal DNA (rDNA) replication origins of Saccharomyces cerevisiae are known to function inefficiently, both in the context of the tandem rDNA repeats in the chromosome and as single copy autonomously replicating sequences (ARSs) in plasmids. Here we examined components of the rDNA ARS that might contribute to inefficient extrachromosomal replication. Like the efficient H4 ARS, the rDNA ARS requires a match to the 11 bp ARS consensus sequence (ACS) and a broad non-conserved region that may contain multiple elements, including a DNA unwinding element (DUE). Using a single-strand-specific nuclease hypersensitivity assay and by determining the superhelical density required for stable DNA unwinding, we found that the DNA of the rDNA ARS is not as easily unwound as the H4 ARS. Unwinding of the rDNA ARS required additional energy, similar to the unwinding of mutations in the H4 ARS that stabilize the double helix in the DUE region and impair replication. In vivo extrachromosomal replication of the rDNA ARS was cold sensitive, like H4 ARS mutants that require additional energy to unwind the DUE region but unlike the easily unwound, wild-type H4 ARS. Impairment of replication function at reduced temperature suggests that the elevated energy requirement for DNA unwinding inherent in the wild-type rDNA ARS contributes to inefficient replication function. We also examined the essential ACS match in the rDNA ARS, which is known to be imperfect at one position. A point mutation in the essential ACS that corrects the imperfect match increased the efficiency of extrachromosomal replication. Our results reveal that the essential ACS element and DNA unwinding in the rDNA ARS are naturally impaired, suggesting that inefficient function of the rDNA replication origin has a biological purpose.     

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 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.     

Functional conservation of multiple elements in yeast chromosomal replicators.

Replicators that control the initiation of DNA replication in the chromosomes of Saccharomyces cerevisiae retain their function when cloned into plasmids, where they are commonly referred to as autonomously replicating sequences (ARSs). Previous studies of the structure of ARS1 in both plasmid and chromosome contexts have shown that it contains one essential DNA element, A, that includes a match to the ARS consensus sequence (ACS), and three additional elements, B1, B2, and B3, that are also important for ARS function. Elements A and B3 are bound by a candidate initiator protein called the origin recognition complex and ARS-binding factor 1, respectively. Although the A and B3 elements have been found in other ARSs, sequence comparisons among ARSs have failed to identify B1- and B2-like elements. To assess the generality of the modular nature of yeast replicators, linker substitution mutagenesis of another yeast chromosomal replicator, ARS307, was performed. Three DNA sequence elements were identified in ARS307, and they were demonstrated to be functionally equivalent to the A, B1, and B2 elements present in ARS1. Despite the lack of DNA sequence similarity, the B1 and B2 elements at each ARS were functionally conserved. Single-base substitutions in the core of the ARS1 B1 and B2 elements identified critical nucleotides required for the function of the B1 element. In contrast, no single-point mutations were found to affect B2 function. The results suggest that multiple DNA sequence elements might be a general and conserved feature of replicator sequences in S. cerevisiae.     

cis-acting components in the replication origin from ribosomal DNA of Saccharomyces cerevisiae.

The ribosomal DNA (rDNA) repeats of Saccharomyces cerevisiae contain an autonomously replicating sequence (ARS) that colocalizes with a chromosomal origin of replication. We show that a minimal sequence necessary for full ARS function corresponds to a 107-bp rDNA fragment which contains three 10-of-11-bp matches to the ARS consensus sequence. Point mutations in only one of the 10-of-11-bp matches, GTTTAT GTTTT, inactivate the rDNA ARS, indicating that this consensus sequence is essential. A perfect match to a revised ARS consensus is present but not essential. Sequences up to 9 bp 5' from the essential consensus are dispensable. A broad DNA region directly 3' to the essential consensus is required and is easily unwound as indicated by: (i) hypersensitivity to nicking of an approximately 100-bp region by mung bean nuclease in a negatively supercoiled plasmid and (ii) helical instability determined by thermodynamic analysis of the nucleotide sequence. A correlation between DNA helical instability and replication efficiency of wild-type and mutated ribosomal ARS derivatives suggests that a broad region 3' to the essential ARS consensus functions as a DNA unwinding element. Certain point mutations that do not stabilize the DNA helix in the 3' region but reduce ARS efficiency reveal an element distinct from, but overlapping, the DNA unwinding element. The nucleotide sequence of the functionally important constituents in the ARS appears to be conserved among the rDNA repeats in the chromosome.     

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.     

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.     

A DNA unwinding element and an ARS consensus comprise a replication origin within a yeast chromosome.

We have defined a replication origin, ORI305, within chromosome III of Saccharomyces cerevisiae by means of mutational analysis. cis-acting elements required for origin activity in the chromosome, as assayed by two-dimensional gel electrophoresis of replication intermediates, are the same as those required for the function of an autonomously replicating sequence, ARS305, in a plasmid. Essential elements include (i) an 11 bp sequence that is a near match to the ARS consensus and (ii) a broad sequence directly 3' to the consensus near match. Origin function is inactivated by point mutations in the essential near match sequence, suggesting that the sequence contributes to specifying the origin in the chromosome. Other consensus near matches with different sequences are present but are not required. The essential 3'-flanking sequence exhibits DNA helical instability and is sensitive to deletion mutations that stabilize the DNA helix. The wild-type 3'-flanking sequence can be functionally substituted by dissimilar sequences that also exhibit helical instability. The requirement for DNA helical instability indicates that the essential 3'-flanking sequence serves as a DNA unwinding element in the chromosome.     

Control of ATP-Dependent Binding of Saccharomyces cerevisiae Origin Recognition Complex to Autonomously Replicating DNA Sequences

Eukaryotic origin recognition complexes (ORCs) play pivotal roles in the initiation of chromosomal DNA replication. ORC from the yeast, Saccharomyces cerevisiae, recognizes and binds replication origins in the late G1 phase and the binding has profound implications in the progression of the cell cycle to the S-phase. Therefore, we have quantitatively analyzed the mechanism of recognition and interaction of the yeast ORC with various elements of a yeast origin of DNA replication, the autonomously replicating sequence 1 (ARS1). ORC bound all four individual A and B elements of ARS1 with reasonably high affinities. However, the highest affinity binding was observed with a DNA sequence containing both the A and B1 elements. In addition, ATP and ADP significantly modulated the binding of ORC to the combined elements as well as modulating the binding of ORC to the element A alone or in combination with the B1 element. However, binding of ORC to individual B1, B2, and B3 elements was not responsive to nucleotides. Thus, the consensus ARS sequence in element A appeared to play a pivotal role in the ATP-dependent binding of ORC to ARS1 and likely in other ARSs or origins of DNA replication.     

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.     

Domain B of ARS307 contains two functional elements and contributes to chromosomal replication origin function.

ARS307 is highly active as a replication origin in its native location on chromosome III of Saccharomyces cerevisiae. Its ability to confer autonomous replication activity on plasmids requires the presence of an 11-bp autonomously replicating sequence (ARS) consensus sequence (ACS), which is also required for chromosomal origin function, as well as approximately 100 bp of sequence flanking the ACS called domain B. To further define the sequences required for ARS function, a linker substitution mutagenesis of domain B was carried out. The mutations defined two sequences, B1 and B2, that contribute to ARS activity. Therefore, like ARS1, domain B of ARS307 is composed of functional subdomains. Constructs carrying mutations in the B1 element were used to replace the chromosomal copy of ARS307. These mutations caused a reduction in chromosomal origin activity, demonstrating that the B1 element is required for efficient chromosomal origin function.     

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.     

Highly organized DnaA-oriC complexes recruit the single-stranded DNA for replication initiation

In Escherichia coli, the replication origin oriC consists of two functional regions: the duplex unwinding element (DUE) and its flanking DnaA-assembly region (DAR). ATP-DnaA molecules multimerize on DAR, unwinding DUE for DnaB helicase loading. However, DUE-unwinding mechanisms and functional structures in DnaA-oriC complexes supporting those remain unclear. Here, using various in vitro reconstituted systems, we identify functionally distinct DnaA sub-complexes formed on DAR and reveal novel mechanisms in DUE unwinding. The DUE-flanking left-half DAR carrying high-affinity DnaA box R1 and the ATP-DnaA-preferential DnaA box R5, τ1-2 and I1-2 sites formed a DnaA sub-complex competent in DUE unwinding and ssDUE binding, thereby supporting basal DnaB loading activity. This sub-complex is further subdivided into two; the DUE-distal DnaA sub-complex formed on the ATP-DnaA-preferential sites binds ssDUE. Notably, the DUE-flanking, DnaA box R1-DnaA sub-complex recruits DUE to the DUE-distal DnaA sub-complex in concert with a DNA-bending nucleoid protein IHF, thereby promoting DUE unwinding and binding of ssDUE. The right-half DAR-DnaA sub-complex stimulated DnaB loading, consistent with in vivo analyses. Similar features are seen in DUE unwinding of the hyperthermophile, Thermotoga maritima, indicating evolutional conservation of those mechanisms.     

High-resolution analysis of four efficient yeast replication origins reveals new insights into the ORC and putative MCM binding elements

In budding yeast, the eukaryotic initiator protein ORC (origin recognition complex) binds to a bipartite sequence consisting of an 11 bp ACS element and an adjacent B1 element. However, the genome contains many more matches to this consensus than actually bind ORC or function as origins in vivo. Although ORC-dependent loading of the replicative MCM helicase at origins is enhanced by a distal B2 element, less is known about this element. Here, we analyzed four highly active origins (ARS309, ARS319, ARS606 and ARS607) by linker scanning mutagenesis and found that sequences adjacent to the ACS contributed substantially to origin activity and ORC binding. Using the sequences of four additional B2 elements we generated a B2 multiple sequence alignment and identified a shared, degenerate 8 bp sequence that was enriched within 228 known origins. In addition, our high-resolution analysis revealed that not all origins exist within nucleosome free regions: a class of Sir2-regulated origins has a stably positioned nucleosome overlapping or near B2. This study illustrates the conserved yet flexible nature of yeast origin architecture to promote ORC binding and origin activity, and helps explain why a strong match to the ORC binding site is insufficient to identify origins within the genome.     

DNA sequence and functional analysis of homologous ARS elements of Saccharomyces cerevisiae and S. carlsbergensis.

ARS elements of Saccharomyces cerevisiae are the cis-acting sequences required for the initiation of chromosomal DNA replication. Comparisons of the DNA sequences of unrelated ARS elements from different regions of the genome have revealed no significant DNA sequence conservation. We have compared the sequences of seven pairs of homologous ARS elements from two Saccharomyces species, S. cerevisiae and S. carlsbergensis. In all but one case, the ARS308-ARS308(carl) pair, significant blocks of homology were detected. In the cases of ARS305, ARS307, and ARS309, previously identified functional elements were found to be conserved in their S. carlsbergensis homologs. Mutation of the conserved sequences in the S. carlsbergensis ARS elements revealed that the homologous sequences are required for function. These observations suggested that the sequences important for ARS function would be conserved in other ARS elements. Sequence comparisons aided in the identification of the essential matches to the ARS consensus sequence (ACS) of ARS304, ARS306, and ARS310(carl), though not of ARS310.     

The ARS consensus sequence is required for chromosomal origin function in Saccharomyces cerevisiae.

Replication origins have been mapped to positions that coincide, within experimental error (several hundred base pairs), with ARS elements. To determine whether the DNA sequences required for ARS function on plasmids are required for chromosomal origin function, the chromosomal copy of ARS306 was deleted and the chromosomal copy of ARS307 was replaced with mutant derivatives of ARS307 containing single point mutations in domain A within the ARS core consensus sequence. The chromosomal origin function of these derivatives was assayed by two-dimensional agarose gel electrophoresis. Deletion of ARS306 deleted the associated replication origin. The effects on chromosomal origin function of mutations in domain A paralleled their effects on ARS function, as measured by plasmid stability. These results demonstrate that chromosomal origin function is a property of the ARS element itself.     

Modular sequence elements associated with origin regions in eukaryotic chromosomal DNA.

We have postulated that chromosomal replication origin regions in eukaryotes have in common clusters of certain modular sequence elements (Benbow, Zhao, and Larson, BioEssays 14, 661-670, 1992). In this study, computer analyses of DNA sequences from six origin regions showed that each contained one or more potential initiation regions consisting of a putative DUE (DNA unwinding element) aligned with clusters of SAR (scaffold associated region), and ARS (autonomously replicating sequence) consensus sequences, and pyrimidine tracts. The replication origins analyzed were from the following loci: Tetrahymena thermophila macronuclear rDNA gene, Chinese hamster ovary dihydrofolate reductase amplicon, human c-myc proto-oncogene, chicken histone H5 gene, Drosophila melanogaster chorion gene cluster on the third chromosome, and Chinese hamster ovary rhodopsin gene. The locations of putative initiation regions identified by the computer analyses were compared with published data obtained using diverse methods to map initiation sites. For at least four loci, the potential initiation regions identified by sequence analysis aligned with previously mapped initiation events. A consensus DNA sequence, WAWTTDDWWWDHWGWHMAWTT, was found within the potential initiation regions in every case. An additional 35 kb of combined flanking sequences from the six loci were also analyzed, but no additional copies of this consensus sequence were found.