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.     

Origin recognition complex binding to a metazoan replication origin

The initiation of DNA replication in eukaryotic cells at the onset of S phase requires the origin recognition complex (ORC) [1]. This six-subunit complex, first isolated in Saccharomyces cerevisiae [2], is evolutionarily conserved [1]. ORC participates in the formation of the prereplicative complex [3], which is necessary to establish replication competence. The ORC-DNA interaction is well established for autonomously replicating sequence (ARS) elements in yeast in which the ARS consensus sequence [4] (ACS) constitutes part of the ORC binding site [2, 5]. Little is known about the ORC-DNA interaction in metazoa. For the Drosophila chorion locus, it has been suggested that ORC binding is dispersed [6]. We have analyzed the amplification origin (ori) II/9A of the fly, Sciara coprophila. We identified a distinct 80-base pair (bp) ORC binding site and mapped the replication start site located adjacent to it. The binding of ORC to this 80-bp core region is ATP dependent and is necessary to establish further interaction with an additional 65-bp of DNA. This is the first time that both the ORC binding site and the replication start site have been identified in a metazoan amplification origin. Thus, our findings extend the paradigm from yeast ARS1 to multicellular eukaryotes, implicating ORC as a determinant of the position of replication initiation.     

Identification of the sequences required for chromosomal replicator function in Kluyveromyces lactis

The analysis of replication intermediates of a Kluyveromyces lactis chromosomal autonomous replicating sequence (ARS), KARS101, has shown that it is active as a chromosomal replicator. KARS101 contains a 50 bp sequence conserved in two other K. lactis ARS elements. The deletion of the conserved sequence in KARS101 completely abolished replicator activity, in both the plasmids and the chromosome. Gel shift assays indicated that this sequence binds proteins present in K. lactis nuclear extracts, and a 40 bp sequence, previously defined as the core essential for K. lactis ARS function, is required for efficient binding. Reminiscent of the origin replication complex (ORC), the binding appears to be ATP dependent. A similar pattern of protection of the core was seen with in vitro footprinting. KARS101 also functions as an ARS sequence in Saccharomyces cerevisiae. A comparative study using S. cerevisiae nuclear extracts revealed that the sequence required for binding is a dodecanucleotide related to the S. cerevisiae ARS consensus sequence and essential for S. cerevisiae ARS activity.     

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.     

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 at intervals of approximately 40 kb. However, both measurements of replication fork rate and studies of hypomorphic alleles of genes encoding replication initiation proteins suggest the question of whether replication origins are more closely spaced than should be required. We approached this question by systematically deleting replicators from chromosome III. The first significant increase in loss rate detected for the 315-kb full-length chromosome occurred only after all five efficient chromosomal replicators in the left two-thirds of the chromosome (ARS305, ARS306, ARS307, ARS309, and ARS310) had been deleted. The removal of the inefficient replicator ARS308 from this originless region caused little or no additional increase in loss rate. Chromosome fragmentations that removed the normally inactive replicators on the left end of the chromosome or the replicators distal to ARS310 on the right arm showed that both groups of replicators contribute significantly to the maintenance of the originless chromosome. Surprisingly, a 142-kb derivative of chromosome III, lacking all sequences that function as autonomously replicating sequence elements in plasmids, replicated and segregated properly 97% of the time. Both the replication initiation protein ORC and telomeres or a linear topology were required for the maintenance of chromosome fragments lacking replicators.     

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.     

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.     

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.     

DNA elements modulating the <Emphasis Type="Italic">KARS12</Emphasis> chromosomal replicator in <Emphasis Type="Italic">Kluyveromyces lactis</Emphasis>

Eukaryotic chromosomal DNA replication is initiated by a highly conserved set of proteins that interact with cis-acting elements on chromosomes called replicators. Despite the conservation of replication initiation proteins, replicator sequences show little similarity from species to species in the small number of organisms that have been examined. Examination of replicators in other species is likely to reveal common features of replicators. We have examined a Kluyeromyces lactis replicator, KARS12, that functions as origin of DNA replication on plasmids and in the chromosome. It contains a 50-bp region with similarity to two other K. lactis replicators, KARS101 and the pKD1 replication origin. Replacement of the 50-bp sequence with an EcoRI site completely abrogated the ability of KARS12 to support plasmid and chromosomal DNA replication origin activity, demonstrating this sequence is a common feature of K. lactis replicators and is essential for function, possibly as the initiator protein binding site. Additional sequences up to 1 kb in length are required for efficient KARS12 function. Within these sequences are a binding site for a global regulator, Abf1p, and a region of bent DNA, both of which contribute to the activity of KARS12. These elements may facilitate protein binding, protein/protein interaction and/or nucleosome positioning as has been proposed for other eukaryotic origins of DNA replication.     

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.     

Sequence requirements for function of the Drosophila chorion gene locus ACE3 replicator and ori-beta origin elements

The developmentally regulated amplification of the Drosophila third chromosome chorion gene locus requires multiple chromosomal elements. Amplification control element third chromosome (ACE3) appears to function as a replicator, in that it is required in cis for the activity of nearby DNA replication origin(s). Ori-beta is the major origin in the locus, and is a sequence-specific element that is sufficient for high-level amplification in combination with ACE3. Sequence requirements for amplification were examined using a transgenic construct that was buffered from chromosomal position effects by flanking insulator elements. The parent construct supported 18- to 20-fold amplification, and contained the 320 bp ACE3, the approximately 1.2 kb S18 chorion gene and the 840 bp ori-beta. Deletion mapping of ACE3 revealed that an evolutionarily conserved 142 bp core sequence functions in amplification in this context. Several deletions had quantitative effects, suggesting that multiple, partially redundant elements comprise ACE3. S. cerevisiae ARS1 origin sequences could not substitute for ori-beta, thereby confirming the sequence specificity of ori-beta. Deletion mapping of ori-beta identified two required components: a 140 bp 5' element and a 226 bp A/T-rich 3' element called the beta-region that has significant homology to ACE3. Antibody to the origin recognition complex subunit 2 (ORC2) recognizes large foci that localize to the endogenous chorion gene loci and to active transgenic constructs at the beginning of amplification. Mutations in Orc2 itself, or the amplification trans regulator satin eliminated the ORC2 foci. By contrast, with a null mutation of chiffon (dbf4-like) that eliminates amplification, diffuse ORC2 staining was still present, but failed to localize into foci. The data suggest a novel function for the Dbf4-like chiffon protein in ORC localization. Chromosomal position effects that eliminated amplification of transgenic constructs also eliminated foci formation. However, use of the buffered vector allowed amplification of transgenic constructs to occur in the absence of detectable foci formation. Taken together, the data suggest a model in which ACE3 and ori-beta nucleate the formation of a ORC2-containing chromatin structure that spreads along the chromosome in a mechanism dependent upon chiffon.     

Differential targeting of Tetrahymena ORC to ribosomal DNA and non‐rDNA replication origins

The Tetrahymena thermophila origin recognition complex (ORC) contains an integral RNA subunit, 26T RNA, which confers specificity to the amplified ribosomal DNA (rDNA) origin by base pairing with an essential cis‐acting replication determinant—the type I element. Using a plasmid maintenance assay, we identified a 6.7 kb non‐rDNA fragment containing two closely associated replicators, ARS1‐A (0.8 kb) and ARS1‐B (1.2 kb). Both replicators lack type I elements and hence complementarity to 26T RNA, suggesting that ORC is recruited to these sites by an RNA‐independent mechanism. Consistent with this prediction, although ORC associated exclusively with origin sequences in the 21 kb rDNA minichromosome, the interaction between ORC and the non‐rDNA ARS1 chromosome changed across the cell cycle. In G₂ phase, ORC bound to all tested sequences in a 60 kb interval spanning ARS1‐A/B. Remarkably, ORC and Mcm6 associated with just the ARS1‐A replicator in G₁ phase when pre‐replicative complexes assemble. We propose that ORC is stochastically deposited onto newly replicated non‐rDNA chromosomes and subsequently targeted to preferred initiation sites prior to the next S phase.     

A role for a replicator dominance mechanism in silencing.

The role of the natural HMR-E silencer in modulating replication initiation and silencing by the origin recognition complex (ORC) was examined. When natural HMR-E was the only silencer controlling HMR, the silencer's ORC-binding site (ACS) was dispensable for replication initiation but essential for silencing, indicating that a non-silencer chromosomal replicator(s) existed in close proximity to the silencer. Further analysis revealed that regions flanking both sides of HMR-E contained replicators. In contrast to replication initiation by the intact silencer, initiation by the non-silencer replicator(s) was abolished in an orc2-1 mutant, indicating that these replicators were extremely sensitive to defects in ORC. Remarkably, the activity of one of the non-silencer replicators correlated with reduced silencing; inactivation of these replicators caused by either the orc2-1 mutation or the deletion of flanking sequences enhanced silencing. These data were consistent with a role for the ORC bound to the HMR-E silencer ACS in suppressing the function of neighboring ORC molecules capable of inhibiting silencing, and indicated that differences in ORC-binding sites within HMR itself had profound effects on ORC function. Moreover, replication initiation by natural HMR-E was inefficient, suggesting that closely spaced replicators within HMR contributed to an inhibition of replication initiation.     

Chromosome stability in saccharomycete yeasts

Mutants with high instability of chromosome III designated Chl+ (chromosome loss) were obtained after irradiation with UV the Z4221-3c1 haploid disomic for chromosome III. The Chl+ mutants can be divided into two classes: 1) CL2, CL3, CL7, CL9, CL11, CL12, CL13 with elevated level of spontaneous inter- and intragenic recombination; 2) CL4, CL8 which unstable maintenance of chromosome III not accompanied with elevation of mitotic recombination frequency. The CL4 and CL8 mutants also reveal, in contrast to other mutants, unstable maintenance of artificial mini-chromosomes with chromosomal replicator ARS1 and centromeric loci CEN3, CEN4, CEN5, CEN6, CEN11. Substitution of ARS1 for other yeast replicators (ARS2, ARS of 2 micron plasmid) leads to no stabilization of mini-chromosomes in mutants. The noncentromeric plasmids containing homologous replicator (or replicators) from Candida maltosa are maintained with the same frequency both in wild type and in mutants. So, the stability of mini-chromosomes in CL4 and CL8 is not connected with uneffective replication of these chromosomes. Instability of chromosome III and mini-chromosomes in CL4 and CL8 is controlled by two nonallelic genes designated chl14 and chl18. We suppose that these genes control the process of centromere interaction with mitotic spindle microtubules.     

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.     

The spatial arrangement of ORC binding modules determines the functionality of replication origins in budding yeast

In the quest to define autonomously replicating sequences (ARSs) in eukaryotic cells, an ARS consensus sequence (ACS) has emerged for budding yeast. This ACS is recognized by the replication initiator, the origin recognition complex (ORC). However, not every match to the ACS constitutes a replication origin. Here, we investigated the requirements for ORC binding to origins that carry multiple, redundant ACSs, such as ARS603. Previous studies raised the possibility that these ACSs function as individual ORC binding sites. Detailed mutational analysis of the two ACSs in ARS603 revealed that they function in concert and give rise to an initiation pattern compatible with a single bipartite ORC binding site. Consistent with this notion, deletion of one base pair between the ACS matches abolished ORC binding at ARS603. Importantly, loss of ORC binding in vitro correlated with the loss of ARS activity in vivo. Our results argue that replication origins in yeast are in general comprised of bipartite ORC binding sites that cannot function in random alignment but must conform to a configuration that permits ORC binding. These requirements help to explain why only a limited number of ACS matches in the yeast genome qualify as ORC binding sites.     

DNA sequence analysis of ARS elements from chromosome III of Saccharomyces cerevisiae: identification of a new conserved sequence.

Four fragments of Saccharomyces cerevisiae chromosome III DNA which carry ARS elements have been sequenced. Each fragment contains multiple copies of sequences that have at least 10 out of 11 bases of homology to a previously reported 11 bp core consensus sequence. A survey of these new ARS sequences and previously reported sequences revealed the presence of an additional 11 bp conserved element located on the 3' side of the T-rich strand of the core consensus. Subcloning analysis as well as deletion and transposon insertion mutagenesis of ARS fragments support a role for 3' conserved sequence in promoting ARS activity.     

Clustered Adenine/Thymine Stretches Are Essential for Function of a Fission Yeast Replication Origin

We have determined functional elements required for autonomous replication of the Schizosaccharomyces pombe ars2004 that acts as an intrinsic chromosomal replication origin. Internal deletion analysis of a 940-bp fragment (ars2004M) showed three regions, I to III, to be required for autonomously replicating sequence (ARS) activity. Eight-base-pair substitutions in the 40-bp region I, composed of arrays of adenines on a DNA strand, resulted in a great reduction of ARS activity. Substitutions of region I with synthetic sequences showed that no specific sequence but rather repeats of three or more consecutive adenines or thymines, without interruption by guanine or cytosine, are required for the ARS activity. The 65-bp region III contains 11 repeats of the AAAAT sequence, while the 165-bp region II has short adenine or thymine stretches and a guanine- and cytosine-rich region which enhances ARS activity. All three regions in ars2004M can be replaced with 40-bp poly(dA/dT) fragments without reduction of ARS activity. Although spacer regions in the ars2004M enhance ARS activity, all could be deleted when an 40-bp poly(dA/dT) fragment was added in place of region I. Our results suggest that the origin activity of fission yeast replicators depends on the number of adenine/thymine stretches, the extent of their clustering, and presence of certain replication-enhancing elements.