Autonomously Replicating Sequence Research Articles

Dpb11 controls the association between DNA polymerases alpha and epsilon and the autonomously replicating sequence region of budding yeast.

Dpb11 is required for chromosomal DNA replication and the S-phase checkpoint in Saccharomyces cerevisiae. Here, we report detection of a physical complex containing Dpb11 and DNA polymerase epsilon (Dpb11-Polepsilon complex). During the S phase of the cell cycle, Dpb11 associated preferentially with DNA fragments containing autonomously replicating sequences (ARSs), at the same time as Polepsilon associated with these fragments. Association of Dpb11 and Polepsilon with these fragments was mutually dependent, suggesting that the Dpb11-Polepsilon complex associates with the ARS. Moreover, Dpb11 was required for the association of Polalpha-primase with the fragments. Thus, it seems likely that association of the Dpb11-Polepsilon complex with the ARS fragments is required for the association of the Polalpha-primase complex. Hydroxyurea inhibits late-origin firing in S. cerevisiae, and the checkpoint genes, RAD53 and MEC1, are involved in this inhibition. In the presence of hydroxyurea at temperatures permissive for cell growth, Polepsilon in dpb11-1 cells associated with early- and late-origin fragments. In wild-type cells, however, it associated only with early-origin fragments. This indicates that Dpb11 may also be involved in the regulation of late-origin firing. Overall, these results suggest that Dpb11 controls the association between DNA polymerases alpha and epsilon and the ARS.

Is there Replication-associated Mutational Pressure in the Saccharomyces cerevisiae Genome?

Compositional bias of yeast chromosomes was analysed using detrended DNA walks. Unlike eubacterial chromosomes, the yeast chromosomes did not show the specific asymmetry correlated with origin and terminus of replication. It is probably a result of a relative excess of autonomously replicating sequences (ARS) and of random choice of these sequences in each replication cycle. Nevertheless, the last ARS from both ends of chromosomes are responsible for unidirectional replication of subtelomeric sequences with pre-established leading/lagging roles of DNA strands. In these sequences a specific asymmetry is observed, resembling the asymmetry introduced by replication-associated mutational pressure into eubacterial chromosomes.

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.

ORC binding, gene amplification, and the nature of metazoan replication origins.

Genomic DNA replication is a critical process shaped by strong evolutionary pressures. Rapid replication can give a viral or cellular genome an immediate advantage over its competitors as long as requisitely high accuracy is not sacrificed. Prokaryotes employ an extremely fast, accurate DNA replication mechanism utilizing sophisticated multiprotein machines (Kornberg and Baker 1992; Baker and Bell 1998). Genome duplication is controlled by the assembly and activation of bidirectional replication forks at specific replication origins, under the control of initiator proteins that bind origins sequence specifically. For example, Escherichia coli oriC binds multiple copies of the initiator protein, dnaA, at repeated motifs. In an ATP-dependent manner dnaA then locally unwinds the DNA, facilitates helicase loading, and organizes the assembly of polymerases, primases, and other components of the replication fork. Special proteins are required to assemble and maintain this unwieldy machinery. Thus, loading the ring-shaped six-protein dnaB helicase around the DNA requires dnaC, whereas proteins such as tau probably act as scaffolds, organizing replication fork components (including polymerases). Despite the greater complexity of eukaryotic chromosomes, work carried out in Saccharomyces cerevisiae indicates that its genome replicates via a strongly similar mechanism (Leatherwood 1998). Each yeast chromosome contains multiple discrete replication origins where prereplication complexes are assembled during late M and G1 phases. Fully formed, bidirectional replication forks are then activated during S phase. Replication origins can be readily identified as autonomously replicating sequence (ARS) elements that support the propagation of extrachromosomal plasmids. Budding yeast origins are ∼100 bp in size and share an A/T-rich ARS consensus sequence (ACS) that is essential for origin function. Six proteins that form the origin recognition complex (ORC) bind to the ACS in an ATP-dependent manner throughout the cell cycle (Bell and Stillman 1992). Beginning in late mitosis, Cdc6p interacts with origin-bound ORC to bring the six-protein MCM complex, a putative replicative helicase, onto the DNA (Aparicio et al. 1997; Perkins and Diffley 1997; Tanaka et al. 1997). In a late step requiring cyclin-dependent kinase (cdk) activity, Cdc45p is added to the prereplication complex, possibly as a scaffold component (Zou and Stillman 1999). Activation of another origin-associated kinase, Cdc7p, by its regulatory subunit Dbf4p, probably serves as the final signal activating replication fork movement (Dowell et al. 1994; Hardy 1998; Brown and Kelly 1999). By the time prereplication complexes fire, high cdk activity precludes the assembly of new complexes. Because a checkpoint ensures that the cell will not divide until replication is complete, and high levels of cdk activity are not reversed until mitosis is nearly finished, the genome will be replicated once and only once per cell cycle.

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.

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.

Activation of silent replication origins at autonomously replicating sequence elements near the HML locus in budding yeast.

In the budding yeast, Saccharomyces cerevisiae, replicators can function outside the chromosome as autonomously replicating sequence (ARS) elements; however, within chromosome III, certain ARSs near the transcriptionally silent HML locus show no replication origin activity. Two of these ARSs comprise the transcriptional silencers E (ARS301) and I (ARS302). Another, ARS303, resides between HML and the CHA1 gene, and its function is not known. Here we further localized and characterized ARS303 and in the process discovered a new ARS, ARS320. Both ARS303 and ARS320 are competent as chromosomal replication origins since origin activity was seen when they were inserted at a different position in chromosome III. However, at their native locations, where the two ARSs are in a cluster with ARS302, the I silencer, no replication origin activity was detected regardless of yeast mating type, special growth conditions that induce the transcriptionally repressed CHA1 gene, trans-acting mutations that abrogate transcriptional silencing at HML (sir3, orc5), or cis-acting mutations that delete the E and I silencers containing ARS elements. These results suggest that, for the HML ARS cluster (ARS303, ARS320, and ARS302), inactivity of origins is independent of local transcriptional silencing, even though origins and silencers share key cis- and trans-acting components. Surprisingly, deletion of active replication origins located 25 kb (ORI305) and 59 kb (ORI306) away led to detection of replication origin function at the HML ARS cluster, as well as at ARS301, the E silencer. Thus, replication origin silencing at HML ARSs is mediated by active replication origins residing at long distances from HML in the chromosome. The distal active origins are known to fire early in S phase, and we propose that their inactivation delays replication fork arrival at HML, providing additional time for HML ARSs to fire as origins.

Minichromosome Maintenance as a Genetic Assay for Defects in DNA Replication

Minichromosome maintenance (mcm) is an effective genetic assay for mutants defective in DNA replication. Two classes of mcm mutants have been identified using this screen: those that differentially affect the activities of certain autonomously replicating sequences (ARSs) and those that uniformly affect the activities of all ARSs. The ARS-specific MCM genes are essential for the initiation of DNA replication. Among these are members of the MCM2-7 family that encode subunits of the preinitiation complex and MCM10, whose gene product interacts with members of the Mcm2-7 proteins. Among the ARS-nonspecific MCM gene products are chromosome transmission factors. Refinement of this genetic assay as a screening tool and further analysis of existing mcm mutants may reveal new replication initiation proteins.

A dominant selection system designed for copy-number-controlled gene integration in Hansenula polymorpha DL-1.

To facilitate the selection of multiple gene integrants in Hansenula polymorpha, a rapid and copy-number-controlled selection system was developed using a vector containing a telomeric autonomous replication sequence and the bacterial aminoglycoside 3-phosphotransferase (APH) gene. Direct use of the unmodified APH gene as a dominant selectable marker resulted in the extremely slow growth of transformants and the frequent selection of spontaneous resistance. For the proper performance of the APH gene, a set of deleted glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoters of H. polymorpha were fused to the APH gene. The fusion construct with the 578-bp GAPDH promoter conferred G418 resistance sufficient to allow rapid growth of transformants, and thus facilitated the selection of transformants with up to 15 tandem copies of the vector. To increase further the integration copy number within the gene-dose-dependent range, the GAPDH promoter was serially deleted down to the -61 nucleotide. With this weak expression cassette, the integration copy number could easily be controlled between 1 and 50. Tandemly integrated copies of plasmids near the end of the chromosome were mitotically stable over 150 generations. The dosage-dependent selection system of this study would provide a powerful tool for the development of H. polymorpha as an industrial strain to produce recombinant proteins.

Construction of an autonomously replicating plasmid in n-alkane-assimilating yeast, Candida tropicalis

A transformation system using the autonomously replicating plasmid in the n-alkane-assimilating and asporogenic diploid yeast, Candida tropicalis, was developed. For the cloning of a DNA fragment containing a potential autonomously replicating sequence (ARS) from the genomic DNA of C. tropicalis, the ura3 mutant obtained using ethylmethane sulfonate as the host and the URA3 gene amplified by PCR using the C. tropicalis genomic DNA as a selectable marker were prepared. Comparison of ARSs among yeasts revealed that the consensus sequence found in S. cerevisiae was also present in C. tropicalis. The autonomously replicating plasmid containing the putative ARS as the shuttle vector, capable of replicating in both E. coli and C. tropicalis, was first constructed. The transformation system using this plasmid, in addition to the integrative transformation system, will be applicable to genetic studies of C. tropicalis.

MYCDIRE, a dispersed repetitive DNA element in arbuscular mycorrhizal fungi

An EcoRI 1428 bp A + T-rich, DNA element ( MYCDIRE : MYCorrhizal fungus Dispersed Repetitive Element) has been identified in the genome of Scutellospora castanea , an arbuscular mycorrhizal fungus. After sequencing, primers were designed and used in PCR reactions to amplify portions of this element in five species of arbuscular mycorrhizal fungi belonging to four genera of Glomales. Southern hybridizations and partial analysis of the main PCR products confirmed that this element was highly conserved in all the species. Southern blot analysis of single and double digested DNA from two of the glomalean fungi gave smears suggesting that MYCDIRE is scattered throughout the fungal genome. Sequence analyses revealed three copies of a previously reported autonomously replicating sequence ( ARS ) from Saccharomyces cerevisiae and comparisons indicated similarities of MYCDIRE with the autonomously replicating, A + T-rich element ( ACARS ) from Acremonium chrysogenum.

Influence of a replication enhancer on the hierarchy of origin efficiencies within a cluster of DNA replication origins

DNA replication origins in animal cells sometimes occur in clusters. Often one of the multiple origins within these clusters fires more frequently than the others. The reason for this hierarchy remains unknown. Similar origin clusters occur in the fission yeast, Schizosaccharomyces pombe. One such cluster is located near the ura4 gene on chromosome III and contains three origins: ars3002, ars3003, and ars3004. In their natural chromosomal context ( ars3003 is about 2.5 kb upstream of ars3002 and ars3004 is adjacent to ars3002 on the downstream side) their initiation frequencies display a striking hierarchy: ars3002 ⪢ ars3003 ⪢ ars3004. Here, we describe experiments that reveal a 400 bp replication enhancer within ars3004, adjacent to ars3002. The enhancer is essential for ars3004 origin function in a plasmid, but even with the enhancer ars3004 is an inefficient origin. The enhancer is not essential for ars3002 plasmid origin activity, but dramatically stimulates this activity, converting ars3002 from an inefficient plasmid origin to a very efficient one. It also stimulates the plasmid origin activity of ars3001 and ars3003 at all tested positions and orientations on both sides of each autonomously replicating sequence (ARS) element. If ars3002 is redefined to include the enhancer, then the relative activities of the three ARS elements as single origins within separate plasmids or as origins when all three ARS elements are present in a single plasmid is the same as the chromosomal hierarchy. Thus, this replication enhancer defines the relative activities of the three origins in the ura4 origin region. Similar enhancers may affect relative activities in the origin clusters of animal cells.

A role for the yeast SWI/SNF complex in DNA replication.

The yeast SWI/SNF complex is required for expression of many genes and for the full functioning of several transcriptional activators. Genetic and biochemical studies indicate that SWI/SNF uses the energy of ATP hydrolysis to antagonize chromatin-mediated transcriptional repression. We have tested the possibility that SWI/SNF might also play a role in DNA replication. A mitotic minichromosome stability assay was used to investigate the replication efficiency of a variety of autonomous replication sequences (ARSs) in the presence and absence of SWI/SNF. The stability of minichromosomes that contain ARS1, ARS309 or ARS307 is not altered by lack of SWI/SNF, whereas the functioning of ARS121 is crippled when SWI/SNF is inactivated. The SWI/SNF dependence of ARS121 does not require the replication enhancer factor, ABF1, and thus, it appears to be a property of a minimal ARS121 origin. Likewise, a minimal derivative of ARS1 that lacks the ABF1 replication enhancer acquires SWI/SNF dependence. Replacing the ABF1 binding site at ARS1 with a binding site for the LexA-GAL4 chimeric activator also creates a SWI/SNF-dependent ARS. Our studies suggest that the SWI/SNF chromatin remodeling complex can play a role in both replication and transcription and, furthermore, that SWI/SNF dependence of ARS elements is a property of both an ARS-specific replication enhancer and the overall organization of ARS sequence elements.

Chromosomal ARS1 Has a Single Leading Strand Start Site

Initiation sites for DNA synthesis in the chromosomal autonomously replicating sequence (ARS)1 of Saccharomyces cerevisiae were detected at the nucleotide level. The transition from discontinuous to continuous synthesis defines the origin of bidirectional replication (OBR), which mapped adjacent to the origin recognition complex binding site. To ascertain which sites represented starts for leading or lagging strands, we characterized DNA replication from ARS1 in a cdc9 (DNA ligase I) mutant, defective for joining Okazaki fragments. Leading strand synthesis in ARS1 initiated at only a single site, the OBR. Thus, replication in S. cerevisiae is not initiated stochastically by choosing one out of multiple possible sites but, rather, is a highly regulated process with one precise start point.

The autonomously replicating sequence (ARS) of the yeast Saccharomyces exiguus Yp74L-3.

Fragments containing ARSes were cloned from the genomic DNA of the yeast Saccharomyces exiguus Yp74L-3, and the essential regions for ARSes were restricted for these fragments. Mapping studies of ARS-acting sequences in one of these fragments suggested that S. exiguus recognizes a sequence as an ARS that is different from that recognized by Saccharomyces cerevisiae. Two ARS essential regions of S. exiguus were sequenced, and an ARS core consensus sequence of S. exiguus was deduced to be MATTAMWAWWTK. This sequence differs significantly from that of S. cerevisiae in two positions, suggesting that these nucleotide substitutions cause the difference in the ARS-recognition modes between S. exiguus and S. cerevisiae.

A family of telomere-associated autonomously replicating sequences and their functions in targeted recombination in Hansenula polymorpha DL-1.

A family of multiple autonomously replicating sequences (ARSs) which are located at several chromosomal ends of Hansenula polymorpha DL-1 has been identified and characterized. Genomic Southern blotting with an ARS, HARS36, originating from the end of a chromosome, as a probe showed several homologues in the genome of H. polymorpha. Nucleotide sequences of the three fragments obtained by a selective cloning for chromosomal ends were nearly identical to that of HARS36. All three fragments harbored an ARS motif and ended with 18 to 23 identical repetitions of 5'-GGGTGGCG-3' which resemble the telomeric repeat sequence in other eukaryotes. Transformation of H. polymorpha with nonlinearized plasmids containing the newly obtained telomeric ARSs almost exclusively resulted in the targeted integration of a single copy or multiple tandem copies of the plasmid into the chromosomes. The sensitivity to exonuclease Bal31 digestion of the common DNA fragment in all integrants confirmed the telomeric origin of HARS36 homologues, suggesting that several chromosomal ends, if not all of them, consisted of the same ARS motif and highly conserved sequences observed in HARS36. Even though the frequencies of targeted recombination were varied among the ends of the chromosomes, the overall frequency was over 96%. The results suggested that the integration of the plasmids containing telemeric ARSs occurred largely through homologous recombination at the telomeric repeats, which serve as high-frequency recombination targets.

Cyclin B-cdk1 kinase stimulates ORC- and Cdc6-independent steps of semiconservative plasmid replication in yeast nuclear extracts.

Nuclear extracts from Saccharomyces cerevisiae cells synchronized in S phase support the semiconservative replication of supercoiled plasmids in vitro. We examined the dependence of this reaction on the prereplicative complex that assembles at yeast origins and on S-phase kinases that trigger initiation in vivo. We found that replication in nuclear extracts initiates independently of the origin recognition complex (ORC), Cdc6p, and an autonomously replicating sequence (ARS) consensus. Nonetheless, quantitative density gradient analysis showed that S- and M-phase nuclear extracts consistently promote semiconservative DNA replication more efficiently than G1-phase extracts. The observed semiconservative replication is compromised in S-phase nuclear extracts deficient for the Cdk1 kinase (Cdc28p) but not in extracts deficient for the Cdc7p kinase. In a cdc4-1 G1-phase extract, which accumulates high levels of the specific Clb-Cdk1 inhibitor p40(SIC1), very low levels of semiconservative DNA replication were detected. Recombinant Clb5-Cdc28 restores replication in a cdc28-4 S-phase extract yet fails to do so in the cdc4-1 G1-phase extract. In contrast, the addition of recombinant Xenopus CycB-Cdc2, which is not sensitive to inhibition by p40(SIC1), restores efficient replication to both extracts. Our results suggest that in addition to its well-characterized role in regulating the origin-specific prereplication complex, the Clb-Cdk1 complex modulates the efficiency of the replication machinery itself.

The Molecular Biology of Schwanniomyces occidentalis Klocker

ABSTRACT: This review describes the molecular studies of Schwanniomyces occidentalis (Debaryo-myces occidentalis) concerning transformation, genome, gene cloning, gene structure, gene expression and its characteristics to application. Schw. occidentalis appears to have at least five or seven chromosomes and no native plasmid from the yeast has been reported. Four transformation systems based on complement of Schw. occidentalis auxotrophic mutants were established. Vectors with the replicon of 2-µm plasmid and autonomous replication sequences (ARS) of Saccharomyces cerevisiae and Schw. occidentalis ARS replicated extrachromosomally in Schw. occidentalis transformants, without modification of the transformed vector DNA. So far, at least 21 Schw. occidentalis genes encoding 14 different proteins have been cloned. Most of the Schw. occidentalis genes have shown homologies (45 to 91%) with the corresponding genes of other organisms, especially of S. cerevisiae. However, some Schw. occidentalis genes possess other unique structures for their operators, promoters, transcription initiation sites, and terminators. Some foreign genes were expressed in Schw. occidentalis, wldle Schw. occidentalis genes functioned in other yeasts and bacteria, Escherichia coli, and Streptomyces lividans. Due to a strong ability of secretion and low level of glycosylation, Schw. occidentalis might be a promising host to produce heterologous proteins.

Overlapping 3'-end formation signals and ARS elements: tightly linked but functionally separable.

3'-End formation signals are closely associated with autonomous replicating sequences (ARSs) in Saccharomyces cerevisiae in that ARSs frequently contain signals that direct 3'-end formation (Chen et al., 1996). Mutationally-inactivated ARSs that co-reside with 3'-end formation sequences do not disrupt 3'-end formation, thus demonstrating that replication function does not affect termination function. To test the corollary possibility that 3'-end formation is important for replication function, we made point mutations in ARS305 that increase readthrough of the 3'-end formation signals and determined plasmid replication efficiency. Replication efficiency, as assessed by plasmid stability assays, was not altered by mutations affecting 3'-end formation when transcription through the ARS was either absent or highly-induced. Under conditions of high-level transcription through the ARS, the rate of plasmid loss in both wild-type and mutated terminators increased over five-fold from rates observed during transcriptionally repressed conditions. This result indicates that the native 3'-end formation signal is incapable of protecting the replication function when high levels of transcription are directed into the ARS. Thus, the compact nature of the S. cerevisiae genome, rather than a functional inter-dependence, may account for close association of transcription terminators and ARSs.

Multiple initiations in the c-myc replication origin independent of chromosomal location.

At supramolecular resolution, DNA synthesis begins at preferred replication origins in the chromosomes of metazoan cells. To characterize one of these origins in detail, the initiation of replication was examined in the HeLa c-myc origin. Polymerase chain reaction (PCR) amplification of size-fractionated nascent chromosomal DNAs revealed multiple replication initiation sites over a 12-kb region spanning the c-myc origin, including the transcribed region and the 5' and 3' flanking DNA of the gene. Two of the start sites for chromosomal replication occurred inside a 2.4-kb region of the origin that exhibits autonomously replicating sequence (ARS) activity. When a plasmid containing the 2.4-kb ARS region was transfected into HeLa cells, PCR mapping of nascent plasmid DNA confirmed that the plasmid replicated semiconservatively and autonomously and that replication did not initiate at random sites but rather began at multiple sites in a limited zone overlapping the c-myc DNA insert. Within the resolution of the PCR assay, the same sites that were used in the chromosomal c-myc origin were used in the 2.4-kb ARS fragment. The locations of replication start sites determined by PCR are considered in the context of other functional and structural elements of the c-myc origin.