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The autonomously replicating sequence (ARS) element ars3002 is associated with the most active replication origin within a cluster of three closely spaced origins on chromosome III of Schizosaccharomyces pombe. A 361-bp portion of ars3002 containing detectable ARS activity includes multiple near matches to the S. pombe ARS consensus sequence previously reported by Maundrell et al. (K. Maundrell, A. Hutchison, and S. Shall, EMBO J. 7:2203–2209, 1988). Using a gel shift assay with a multimer of an oligonucleotide containing three overlapping matches to the Maundrell ARS consensus sequence, we have detected several proteins in S. pombe crude extracts that bind to the oligonucleotide and ars3002. One of these proteins, ARS binding protein 1, was previously described (Abp1 [Y. Murakami, J. A. Huberman, and J. Hurwitz, Proc. Natl. Acad. Sci. USA 93:502–507, 1996]). In this report the isolation, characterization, and cloning of a second binding activity, designated ARS binding protein 2 (Abp2), are described. Purified Abp2 has an apparent molecular mass of 75 kDa. Footprinting analyses revealed that it binds preferentially to overlapping near matches to the Maundrell ARS consensus sequence. The gene abp2 was isolated, sequenced, and overexpressed in Escherichia coli. The DNA binding activity of overexpressed Abp2 was similar to that of native Abp2. The deduced amino acid sequence contains a region similar to a proline-rich motif (GRP) present in several proteins that bind A+T-rich DNA sequences. Replacement of amino acids within this motif with alanine either abolished or markedly reduced the DNA binding activity of the mutated Abp2 protein, indicating that this motif is essential for the DNA binding activity of Abp2. Disruption of the abp2 gene showed that the gene is not essential for cell viability. However, at elevated temperatures the null mutant was less viable than the wild type and exhibited changes in nuclear morphology. The null mutant entered mitosis with delayed kinetics when DNA replication was blocked with hydroxyurea, and advancement through mitosis led to the loss of cell viability and aberrant formation of septa. The null mutant was also sensitive to UV radiation, suggesting that Abp2 may play a role in regulating the cell cycle response to stress signals.

Previous investigations have shown that the fission yeast, Schizosaccharomyces pombe, has DNA replication origins (500 to 1500 bp) that are larger than those in the budding yeast, Saccharomyces cerevisiae (100 to 150 bp). Deletion and linker substitution analyses of two fission yeast origins revealed that they contain multiple important regions with AT-rich asymmetric (abundant A residues in one strand and T residues in the complementary strand) sequence motifs. In this work we present the characterization of a third fission yeast replication origin, ars3001, which is relatively small (∼570 bp) and responsible for replication of ribosomal DNA. Like previously studied fission yeast origins, ars3001 contains multiple important regions. The three most important of these regions resemble each other in several ways: each region is essential for origin function and is at least partially orientation dependent, each region contains similar clusters of A+T-rich asymmetric sequences, and the regions can partially substitute for each other. These observations suggest that ars3001 function requires synergistic interactions between domains binding similar proteins. It is likely that this requirement extends to other fission yeast origins, explaining why such origins are larger than those of budding yeast.

Background

Replication and transcription, the two key functions of DNA, require unwinding of the DNA double helix. It has been shown that replication origins in the budding yeast, Saccharomyces cerevisiae contain an easily unwound stretch of DNA. We have used a recently developed method for determining the locations and degrees of stress-induced duplex destabilization (SIDD) for all the reported replication origins in the genome of the fission yeast, Schizosaccharomyces pombe.

Results

We have found that the origins are more susceptible to SIDD as compared to the non-origin intergenic regions (NOIRs) and genes. SIDD analysis of many known origins in other eukaryotes suggests that SIDD is a common property of replication origins. Interestingly, the previously shown deletion-dependent changes in the activities of the origins of the ura4 origin region on chromosome 3 are paralleled by changes in SIDD properties, suggesting SIDD’s role in origin activity. SIDD profiling following in silico deletions of some origins suggests that many of the closely spaced S. pombe origins could be clusters of two or three weak origins, similar to the ura4 origin region.

Conclusion

SIDD appears to be a highly conserved, functionally important property of replication origins in S. pombe and other organisms. The distinctly low SIDD scores of origins and the long range effects of genetic alterations on SIDD properties provide a unique predictive potential to the SIDD analysis. This could be used in exploring different aspects of structural and functional organization of origins including interactions between closely spaced origins.

Two functionally important DNA sequence elements in centromeres of the fission yeast Schizosaccharomyces pombe are the centromeric central core and the K-type repeat. Both of these DNA elements show internal functional redundancy that is not correlated with a conserved DNA sequence. Specific, but degenerate, sequences in these elements are bound in vitro by the S. pombe DNA-binding proteins Abp1p (also called Cbp1p) and Cbhp, which are related to the mammalian centromere DNA-binding protein CENP-B. In this study, we determined that Abp1p binds to at least one of its target sequences within S. pombe centromere II central core (cc2) DNA with an affinity (Ks = 7 × 109 M−1) higher than those of other known centromere DNA-binding proteins for their cognate targets. In vivo, epitope-tagged Cbhp associated with centromeric K repeat chromatin, as well as with noncentromeric regions. Like abp1+/cbp1+, we found that cbh+ is not essential in fission yeast, but a strain carrying deletions of both genes (Δabp1 Δcbh) is extremely compromised in growth rate and morphology and missegregates chromosomes at very high frequency. The synergism between the two null mutations suggests that these proteins perform redundant functions in S. pombe chromosome segregation. In vitro assays with cell extracts with these proteins depleted allowed the specific assignments of several binding sites for them within cc2 and the K-type repeat. Redundancy observed at the centromere DNA level appears to be reflected at the protein level, as no single member of the CENP-B-related protein family is essential for proper chromosome segregation in fission yeast. The relevance of these findings to mammalian centromeres is discussed.

How early- and late-firing origins are selected on eukaryotic chromosomes is largely unknown. Here, we show that Mrc1, a conserved factor required for stabilization of stalled replication forks, selectively binds to the early-firing origins in a manner independent of Cdc45 and Hsk1 kinase in the fission yeast Schizosaccharomyces pombe. In mrc1Δ cells (and in swi1Δ cells to some extent), efficiency of firing is stimulated, and its timing is advanced selectively at those origins that are normally bound by Mrc1. In contrast, the late or inefficient origins which are not bound by Mrc1 are not activated in mrc1Δ cells. The enhanced firing and precocious Cdc45 loading at Mrc1-bound early-firing origins are not observed in a checkpoint mutant of mrc1, suggesting that non-checkpoint function is involved in maintaining the normal program of early-firing origins. We propose that prefiring binding of Mrc1 is an important marker of early-firing origins which are precociously activated by the absence of this protein.

The DNA requirements for centromere function in fission yeast have been investigated using a minichromosome assay system. Critical elements of Schizosaccharomyces pombe centromeric DNA are portions of the centromeric central core and sequences within a 2.1-kilobase segment found on all three chromosomes as part of the K-type (K/K"/dg) centromeric repeat. The S. pombe centromeric central core contains DNA sequences that appear functionally redundant, and the inverted repeat motif that flanks the central core in all native fission yeast centromeres is not essential for centromere function in circular minichromosomes. Tandem copies of centromeric repeat K", in conjunction with the central core, exert an additive effect on centromere function, increasing minichromosome mitotic stability with each additional copy. Centromeric repeats B and L, however, and parts of the central core and its core-associated repeat are dispensable and cannot substitute for K-type sequences. Several specific protein binding sites have been identified within the centromeric K-type repeat, consistent with a recently proposed model for centromere/kinetochore function in S. pombe.

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We characterized a number of widely used yeast-Escherichia coli shuttle vectors in the fission yeast Schizosaccharomyces pombe. The 2 micron vectors pDB248 and YEp13 showed high frequency of transformation, intermediate mitotic and low meiotic stability, and a low copy number in S. pombe, analogous to their behavior in [cir0] strains of Saccharomyces cerevisiae. The S. cerevisiae integration vectors pLEU2 and pURA3 transformed S. pombe at very low frequencies but, surprisingly, in a nonintegrative fashion. Instead, they replicated autonomously, and they showed very high copy numbers (up to 150 copies per plasmid-containing cell). This could reflect a lack of sequence specificity for replication of plasmid DNA in S. pombe. pFL20, an S. pombe ars vector, and a series of plasmids derived from it were studied to analyze the unusually high stability of this plasmid. Mitotic stability and partitioning of the plasmids was measured by pedigree analysis of transformed S. pombe cells. An S. pombe DNA fragment (stb) was identified that stabilizes pFL20 by improvement of plasmid partitioning in mitosis and meiosis.

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Background

In budding yeast, the replication checkpoint slows progress through S phase by inhibiting replication origin firing. In mammals, the replication checkpoint inhibits both origin firing and replication fork movement. To find out which strategy is employed in the fission yeast, Schizosaccharomyces pombe, we used microarrays to investigate the use of origins by wild-type and checkpoint-mutant strains in the presence of hydroxyurea (HU), which limits the pool of deoxyribonucleoside triphosphates (dNTPs) and activates the replication checkpoint. The checkpoint-mutant cells carried deletions either of rad3 (which encodes the fission yeast homologue of ATR) or cds1 (which encodes the fission yeast homologue of Chk2).

Results

Our microarray results proved to be largely consistent with those independently obtained and recently published by three other laboratories. However, we were able to reconcile differences between the previous studies regarding the extent to which fission yeast replication origins are affected by the replication checkpoint. We found (consistent with the three previous studies after appropriate interpretation) that, in surprising contrast to budding yeast, most fission yeast origins, including both early- and late-firing origins, are not significantly affected by checkpoint mutations during replication in the presence of HU. A few origins (~3%) behaved like those in budding yeast: they replicated earlier in the checkpoint mutants than in wild type. These were located primarily in the heterochromatic subtelomeric regions of chromosomes 1 and 2. Indeed, the subtelomeric regions defined by the strongest checkpoint restraint correspond precisely to previously mapped subtelomeric heterochromatin. This observation implies that subtelomeric heterochromatin in fission yeast differs from heterochromatin at centromeres, in the mating type region, and in ribosomal DNA, since these regions replicated at least as efficiently in wild-type cells as in checkpoint-mutant cells.

Conclusion

The fact that ~97% of fission yeast replication origins – both early and late – are not significantly affected by replication checkpoint mutations in HU-treated cells suggests that (i) most late-firing origins are restrained from firing in HU-treated cells by at least one checkpoint-independent mechanism, and (ii) checkpoint-dependent slowing of S phase in fission yeast when DNA is damaged may be accomplished primarily by the slowing of replication forks.

We have used micrococcal nuclease (MNase) digestion followed by deep sequencing in order to obtain a higher resolution map than previously available of nucleosome positions in the fission yeast, Schizosaccharomyces pombe. Our data confirm an unusually short average nucleosome repeat length, ∼152 bp, in fission yeast and that transcriptional start sites (TSSs) are associated with nucleosome-depleted regions (NDRs), ordered nucleosome arrays downstream and less regularly spaced upstream nucleosomes. In addition, we found enrichments for associated function in four of eight groups of genes clustered according to chromatin configurations near TSSs. At replication origins, our data revealed asymmetric localization of pre-replication complex (pre-RC) proteins within large NDRs—a feature that is conserved in fission and budding yeast and is therefore likely to be conserved in other eukaryotic organisms.

The polyomavirus origin for DNA replication comprises at least two essential, but functionally distinct, cis-acting components. One of these, the origin core, is required only for DNA replication. It includes binding sites for large T antigen and the origin of bidirectional DNA replication. The other component is required for both transcription and DNA replication and is represented by two functionally redundant regions, alpha and beta, which are elements of the polyomavirus enhancer. Whereas either enhancer element will activate DNA replication, both enhancer elements are required to constitute a functional enhancer of transcription. To identify the sequences that make up each enhancer element, we have subjected them separately to in vitro mutagenesis and measured their capacity to activate replication in cis of the origin core in MOP-8 cells, which provide all trans-acting replicative functions including large T antigen. The results reveal that the beta enhancer element is composed of three subelements, two auxiliary subelements, and a core subelement. The core subelement independently activated DNA replication, albeit poorly. The auxiliary subelements, which were inactive on their own, acted synergistically with the core subelement to increase its activity. Interestingly, dimers of the beta core subelement functioned as well as the combination of a beta auxiliary subelement and a core subelement, suggesting that the subelements are functionally equivalent. The alpha enhancer element is organized similarly; it too comprises an auxiliary subelement and a core subelement. These results lead us to suggest that the polyomavirus enhancer comprises two levels of organization; two or more enhancer elements form an enhancer, and two or more subelements make up an enhancer element. The subelements share few sequences and serve as binding sites for distinct cellular factors. It appears, therefore, that a number of different cellular proteins function cooperatively to activate polyomavirus DNA replication by a common mechanism.

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The feasibility of using the fission yeast, Schizosaccharomyces pombe , as a host for the propagation of cloned large fragments of human DNA has been investigated. Two acentric vector arms were utilized; these carry autonomously replicating sequences ( ars elements), selectable markers ( ura4(+) or LEU2 ) and 250 bp of S. pombe terminal telomeric repeats. All cloning was performed between the unique sites in both vector arms for the restriction endonuclease Not I. Initially the system was tested by converting six previously characterized cosmids from human chromosome 11p13 into a form that could be propagated in S.pombe as linear episomal elements of 50-60 kb in length. In all transformants analysed these cosmids were maintained intact. To test if larger fragments of human DNA could also be propagated total human DNA was digested with Not I and size fractionated by pulsed field gel electrophoresis (PFGE). Fractions of 100-1000 kb were ligated to Not I-digested vector arms and transformed into S.pombe protoplasts in the presence of lipofectin. Prototrophic ura+leu+transformants were obtained which upon examination by PFGE were found to contain additional linear chromosomes migrating at between 100 and 500 kb with a copy number of 5-10 copies/cell. Hybridization analyses revealed that these additional bands contained human DNA. Fluorescent in situ hybridization (FISH) analyses of several independent clones indicated that the inserts were derived from single loci within the human genome. These analyses clearly demonstrate that it is possible to clone large fragments of heterologous DNA in fission yeast using this S.p ombe artificial chromosome system which we have called SPARC. This vector-host system will complement the various other systems for cloning large DNA fragments.

While many of the proteins involved in the initiation of DNA replication are conserved between yeasts and metazoans, the structure of the replication origins themselves has appeared to be different. As typified by ARS1, replication origins in Saccharomyces cerevisiae are <150 bp long and have a simple modular structure, consisting of a single binding site for the origin recognition complex, the replication initiator protein, and one or more accessory sequences. DNA replication initiates from a discrete site. While the important sequences are currently less well defined, metazoan origins appear to be different. These origins are large and appear to be composed of multiple, redundant elements, and replication initiates throughout zones as large as 55 kb. In this report, we characterize two S. cerevisiae replication origins, ARS101 and ARS310, which differ from the paradigm. These origins contain multiple, redundant binding sites for the origin recognition complex. Each binding site must be altered to abolish origin function, while the alteration of a single binding site is sufficient to inactivate ARS1. This redundant structure may be similar to that seen in metazoan origins.

We have examined the feasibility and efficiency of PCR-mediated direct gene disruptions in the fission yeast Schizosaccharomyces pombe. In the present study, the S.pombe ura4+ gene was amplified by PCR with oligonucleotides that had short flanking regions ( approximately 40 bp) to the target gene. Using this purified PCR product we were able to disrupt genes in an S. pombe strain bearing aura4 deletion, with an efficiency ranging between 1 and 3% among selected transformants. The results indicated that despite S.pombe's preference for non-homologous or illegitimate recombination, even very short stretches of homologous regions could be used to target genes at a defined frequency in this organism. The successful disruption of four independent genes (sts1+, gcs1+, gsh2+and hmt1+) by this method further demonstrates that, despite the relatively low efficiency, the method is very feasible, and it's simplicity, especially when coupled to phenotype-based screening, should greatly facilitate disruption of genes in S.pombe.

We isolated four fragments from the Schizosaccharomyces pombe genome that mediate autonomous replication. A two-dimensional gel analysis revealed that in each case initiation could be mapped to within the S. pombe sequences. In three of the fragments, initiation could be mapped to one discrete location. In the fourth fragment, subcloning and two-dimensional gel analysis suggested that two discrete origins of replication were located within 3 kb of each other. When in proximity, usually only one of these origins fired, suggesting origin interference. Two-dimensional gel analysis of the four origin fragments at their genomic locations demonstrated that each is used in the chromosomes, but in only a subset of cells or cell divisions. The S. pombe genome appears to contain many discrete origins, not all of which fire in any given cell and some of which are closely spaced. Not I/Sfi I mapping of the five origins from this and a previous study indicates that they are randomly distributed throughout the genome and appear to be representative of chromosomal origins of replication in this organism. We compare the features of S. pombe replication origins with those of S. cerevisiae and animal cells.

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DNA replication initiates at discrete origins along eukaryotic chromosomes. However, in most organisms, origin firing is not efficient; a specific origin will fire in some but not all cell cycles. This observation raises the question of how individual origins are selected to fire and whether origin firing is globally coordinated to ensure an even distribution of replication initiation across the genome. We have addressed these questions by determining the location of firing origins on individual fission yeast DNA molecules using DNA combing. We show that the firing of replication origins is stochastic, leading to a random distribution of replication initiation. Furthermore, origin firing is independent between cell cycles; there is no epigenetic mechanism causing an origin that fires in one cell cycle to preferentially fire in the next. Thus, the fission yeast strategy for the initiation of replication is different from models of eukaryotic replication that propose coordinated origin firing.

The human papovavirus BK virus contains three 68-base-pair (bp) repeats that act as transcriptional enhancers. An analysis of plasmids containing the BK virus origin revealed that sequences within the 68-bp enhancer are required for DNA replication as well as transcription of the early promoter in COS-1 cells. Origins with a single 68-bp repeat replicated as efficiently as did those with three repeats when transfected into COS-1 cells. Replication did not occur in the absence of enhancer sequences and could not be restored by distal placement of enhancers to enhancerless origins. However, as with simian virus 40, replication in vitro was not dependent on the presence of any enhancer sequences. Deletion analysis showed that replication of BK virus origins was dependent on the presence of the first 21 bp of the enhancer contiguous with the A-T-rich stretch of the origin. This 21-bp element is referred to as the rep element. Although in combination with rep the remaining 47 bp of the enhancer appear to increase replication by two- to fivefold, they alone are not sufficient to support replication. Deletions or insertions in the enhancer which did not alter the rep element had no major effect on replication. Site-directed mutagenesis of the Sp1-like site within the rep element, the NF1 site present in the enhancer, or the NF1 site in adjacent late-side sequences each reduced transcription by two- to fivefold, but had no effect on replication, suggesting that replication and transcription can be uncoupled.

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The replication origins (ORIs) of Schizosaccharomyces pombe, like those in most eukaryotes, are long chromosomal regions localized within A+T-rich domains. Although there is no consensus sequence, the interacting proteins are strongly conserved, suggesting that DNA structure is important for ORI function. We used atomic force microscopy in solution and DNA modelling to study the structural properties of the Spars1 origin. We show that this segment is the least stable of the surrounding DNA (9 kb), and contains regions of intrinsically bent elements (strongly curved and inherently supercoiled DNAs). The pORC-binding site co-maps with a superhelical DNA region, where the spatial arrangement of adenine/thymine stretches may provide the binding substrate. The replication initiation site (RIP) is located within a strongly curved DNA region. On pORC unwinding, this site shifts towards the apex of the curvature, thus potentiating DNA melting there. Our model is entirely consistent with the sequence variability, large size and A+T-richness of ORIs, and also accounts for the multistep nature of the initiation process, the specificity of pORC-binding site(s), and the specific location of RIP. We show that the particular DNA features and dynamic properties identified in Spars1 are present in other eukaryotic origins.

Background

Retrotransposons are transposable elements that proliferate within eukaryotic genomes through a process involving reverse transcription. The numbers of retrotransposons within genomes and differences between closely related species may yield insight into the evolutionary history of the elements. Less is known about the ongoing dynamics of retrotransposons, as analysis of genome sequences will only reveal insertions of retrotransposons that are fixed - or near fixation - in the population or strain from which genetic material has been extracted for sequencing. One pre-requisite for retrotransposition is transcription of the elements. Given their intrinsic sequence redundancy, transcriptome-level analyses of transposable elements are scarce. We have used recently published transcriptome data from the fission yeast Schizosaccharomyces pombe to assess the ability to detect and describe transcriptional activity from Long Terminal Repeat (LTR) retrotransposons. LTR retrotransposons are normally flanked by two LTR sequences. However, the majority of LTR sequences in S. pombe exist as solitary LTRs, i.e. as single terminal repeat sequences not flanking a retrotransposon. Transcriptional activity was analysed for both full-length LTR retrotransposons and solitary LTRs.

Results

Two independent sets of transcriptome data reveal the presence of full-length, polyadenylated transcripts from LTR retrotransposons in S. pombe during growth phase in rich medium. The redundancy of retrotransposon sequences makes it difficult to assess which elements are transcriptionally active, but data strongly indicates that only a subset of the LTR retrotransposons contribute significantly to the detected transcription. A considerable level of reverse strand transcription is also detected. Equal levels of transcriptional activity are observed from both strands of solitary LTR sequences. Transcriptome data collected during meiosis suggests that transcription of solitary LTRs is correlated with the transcription of nearby protein-coding genes.

Conclusions

Presumably, the host organism negatively regulates proliferation of LTR retrotransposons. The finding of considerable transcriptional activity of retrotransposons suggests that part of this regulation is likely to take place at a post-transcriptional level. Alternatively, the transcriptional activity may signify a hitherto unrecognized activity level of retrotransposon proliferation. Our findings underline the usefulness of transcriptome data in elucidating dynamics in retrotransposon transcription.

In fission yeast, as in many organisms, episomally replicating plasmid DNA molecules can be used for a wide variety of applications. However, replicating plasmids described previously are each propagated at a high copy number per cell. Plasmid fission yeast twenty (pFY20) contains the ura4+ gene for positive and negative selection, an origin of replication (ars1 ) and a stability element (stb). Although this plasmid does not have a centromere, it is propagated with a copy number of about two plasmids per haploid genome equivalent and it is transmitted with relatively high fidelity in mitosis and meiosis. This low-copy vector is useful for screens and mutational studies where overexpression (e.g. from high copy plasmids) is undesirable. We therefore constructed multiple partial-digest, size-fractionated, fission yeast genomic DNA libraries in pFY20 and in the cloning vector pBluescript KS+. These libraries have sufficient complexity (average of 2100 genome equivalents each) for saturation screening by complementation, plasmid shuffle or hybridization.

Background

DNA replication initiates at distinct origins in eukaryotic genomes, but the genomic features that define these sites are not well understood.

Results

We have taken a combined experimental and bioinformatic approach to identify and characterize origins of replication in three distantly related fission yeasts: Schizosaccharomyces pombe, Schizosaccharomyces octosporus and Schizosaccharomyces japonicus. Using single-molecule deep sequencing to construct amplification-free high-resolution replication profiles, we located origins and identified sequence motifs that predict origin function. We then mapped nucleosome occupancy by deep sequencing of mononucleosomal DNA from the corresponding species, finding that origins tend to occupy nucleosome-depleted regions.

Conclusions

The sequences that specify origins are evolutionarily plastic, with low complexity nucleosome-excluding sequences functioning in S. pombe and S. octosporus, and binding sites for trans-acting nucleosome-excluding proteins functioning in S. japonicus. Furthermore, chromosome-scale variation in replication timing is conserved independently of origin location and via a mechanism distinct from known heterochromatic effects on origin function. These results are consistent with a model in which origins are simply the nucleosome-depleted regions of the genome with the highest affinity for the origin recognition complex. This approach provides a general strategy for understanding the mechanisms that define DNA replication origins in eukaryotes.

The centromere enhancer is a functionally important DNA region within the Schizosaccharomyces pombe centromeric K-type repeat. We have previously shown that addition of the enhancer and cen2 centromeric central core to a circular minichromosome is sufficient to impart appreciable centromere function. A more detailed analysis of the enhancer shows that it is dispensable for centromere function in a cen1-derived minichromosome containing the central core and the remainder of the K-type repeat, indicating that the critical centromeric K-type repeat, like the central core, is characterized by functional redundancy. The centromeric enhancer is required, however, for a central core-carrying minichromosome to exhibit immediate centromere activity when the circular DNA is introduced via transformation into S. pombe. This immediate activation is probably a consequence of a centromere-targeted epigenetic system that governs the chromatin architecture of the region. Moreover, our studies show that two entirely different DNA sequences, consisting of elements derived from two native centromeres, can display centromere function. An S. pombe CENP-B-like protein, Abp1p/Cbp1p, which is required for proper chromosome segregation in vivo, binds in vitro to sites within and adjacent to the modular centromere enhancer, as well as within the centromeric central cores. These results provide direct evidence in fission yeast of a model, similar to one proposed for mammalian systems, whereby no specific sequence is necessary for centromere function but certain classes of sequences are competent to build the appropriate chromatin foundation upon which the centromere/kinetochore can be formed and activated.

We have identified five autonomously replicating sequences (ARSs) in a 100 kbp region of the Schizosaccharomyces pombe chromosome II. Analyses of replicative intermediates of the chromosome DNA by neutral/neutral two-dimensional gel electrophoresis demonstrated that at least three of these ARS loci operate as chromosomal replication origins. One of the loci,ori2004, was utilized in almost every cell cycle, while the others were used less frequently. The frequency of initiation from the respective chromosomal replication origin was found to be roughly proportional to the efficiency of autonomous replication of the corresponding ARS plasmid. Replication from ori2004 was initiated within a distinct region almost the same as that for replication of the ARS plasmid. These results showed that the ori2004 region of approximately 3 kbp contains all the cis elements essential for initiation of chromosome replication.

Two-dimensional gel electrophoretic replicon mapping techniques were used to identify all functional DNA replication origins and termini in a 26.5-kbp stretch in the left arm of yeast chromosome III. Only one origin was detected; it coincided with an ARS element (ARS306), as have all previously mapped yeast origins. A replication termination region was identified in a 4.3-kbp stretch at the telomere-proximal end of the investigated region, between the origin identified in this paper and the neighboring, previously mapped, ARS305-associated origin (previously called the A6C origin). Termination does not occur at a specific site; instead, it appears to be the consequence of replication forks converging in a stretch of DNA of at least 4.3 kbp.

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The SV40 origin of replication comprises a run of thymine and adenine residues. Integrity of this AT-rich sequence is known to be essential for replication. We set out to study whether or not these elements can work synergistically to sustain replication. Quite surprisingly, additional copies of the AT stretch linked to a functional SV40 ori core dramatically reduce its replication in Cosl cells, probably by creating some physical block. Interestingly, the same inhibiting effect can be observed with the addition in cis of the yeast ARS consensus, which is homologous to the SV40 AT stretch. This modulation is possibly due to the action of cellular factors that recognize either of the two sequences. In fact, we demonstrate the existence of factor(s) in Cosl crude nuclear extracts that in vitro can specifically bind to either of them. Moreover, we show that these sequence-specific factor(s) (MW about 50 kDa), named SOAP, recognize both single (T-rich strand) and double stranded forms of the AT tracts. Binding to single stranded AT stretches can be specifically inhibited by the corresponding duplex form, but not vice versa.

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Retrovirus plus-strand synthesis is primed by a cleavage remnant of the polypurine tract (PPT) region of viral RNA. In this study, we tested replication properties for Moloney murine leukemia viruses with targeted mutations in the PPT and in conserved sequences upstream, as well as for pools of mutants with randomized sequences in these regions. The importance of maintaining some purine residues within the PPT was indicated both by examining the evolution of random PPT pools and from the replication properties of targeted mutants. Although many different PPT sequences could support efficient replication and one mutant that contained two differences in the core PPT was found to replicate as well as the wild type, some sequences in the core PPT clearly conferred advantages over others. Contributions of sequences upstream of the core PPT were examined with deletion mutants. A conserved T-stretch within the upstream sequence was examined in detail and found to be unimportant to helper functions. Evolution of virus pools containing randomized T-stretch sequences demonstrated marked preference for the wild-type sequence in six of its eight positions. These findings demonstrate that maintenance of the T-rich element is more important to viral replication than is maintenance of the core PPT.