Specific initiation at an origin of replication from Schizosaccharomyces pombe.

Using a genetic assay for efficient autonomous replication, we have isolated from Schizosaccharomyces pombe a 6.2-kb fragment which shows the properties expected of an origin of DNA replication in S. pombe. A 2.8-kb subclone of the fragment has the same replication properties. Two-dimensional gel analysis of replication intermediates throughout plasmids carrying the 6.2- or 2.8-kb fragments shows that replication initiates only in a specific region, which can be localized to within several hundred base pairs, in the fragments. This region is also a site of replication initiation in the S. pombe chromosome where the fragments normally reside. These results provide strong evidence that initiation of replication in S. pombe is localized and mediated by specific DNA sequence signals.     

Increased recombination intermediates and homologous integration hot spots at DNA replication origins

We have studied the relationship between DNA replication and recombination in Schizosaccharomyces pombe using two-dimensional gel electrophoresis and functional analysis. Our results indicate that the activation of replication origins (ORIs) during the mitotic cell cycle is associated with the generation of joint DNA molecules between sister chromatids. The frequency of integration by homologous recombination was up to 50-fold higher than the genomic average within a narrow window overlapping the ars1 replication initiation site. The S. pombe rad22Delta, rhp51Delta, and rhp54Delta mutants, deficient in mitotic recombination, activate ORIs very inefficiently and accumulate abnormal replication intermediates. These results focus on the general link between replication and recombination previously found in several systems and suggest a role for recombination in the initiation of eukaryotic DNA replication.     

Replication of centromere II of Schizosaccharomyces pombe.

The centromeric DNAs of Schizosaccharomyces pombe chromosomes resemble those of higher eukaryotes in being large and composed predominantly of repeated sequences. To begin a detailed analysis of the mode of replication of a complex centromere, we examined whether any sequences within S. pombe centromere II (cen2) have the ability to mediate autonomous replication. We found a high density of segments with such activity, including at least eight different regions comprising most of the repeated and unique centromeric DNA elements. A physical mapping analysis using two-dimensional gels showed that autonomous replication initiated within the S. pombe sequences in each plasmid. A two-dimensional gel analysis of replication on the chromosomes revealed that the K and L repeat elements, which occur in multiple copies at all three centromeres and comprise approximately 70% of total centromeric DNA mass in S. pombe, are both sites of replication initiation. In contrast, the unique cen2 central core, which contains multiple segments that can support autonomous replication, appears to be repressed for initiation on the chromosome. We discuss the implications of these findings for our understanding of DNA replication and centromere function.     

Identification and characterization of a complex chromosomal replication origin in<Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

In the budding yeast,S. cerevisiae, two-dimensional (2D) gel electrophoresis techniques permit mapping of DNA replication origins to short stretches of DNA (±300 bp). In contrast, in mammalian cells andDrosophila, 2D gel techniques do not permit precise origin localization; the results have been interpreted to suggest that replication initiates in broad zones (several kbp or more). However, alternative techniques (replication timing, nascent strand polarity analysis, nascent strand size analysis) suggest that mammalian origins can be mapped to short DNA stretches, just likeS. cerevisiae origins. Because the fission yeast,Schizosaccharomyces pombe, resembles higher organisms in several ways to a greater extent than doesS. cerevisiae, we thought thatS. pombe replication origins might prove to resemble — and thus be helpful models for — animal cell origins. An attempt to test this possibility using 2D gel techiques resulted in identification of a replication origin near theura4 gene on chromosome III ofS. pombe. The 2D gel patterns produced by thisS. pombe origin indeed resemble the patterns produced by animal cell origins and show that theS. pombe origin cannot be precisely located. The data suggest an initiation zone of 3–5 kbp. Some aspects of the 2D gel patterns detected at theS. pombe origin cannot be explained by the rationale of initiation in broad zones, suggesting that future biochemical and genetic studies of this complex origin are likely to provide information useful in helping to understand the apparent conflict between the 2D gel mapping techniques and other mapping techniques at animal cell origins.     

Origins and complexes: the initiation of DNA replication

Eukaryotic DNA is organized for replication as multiple replicons. DNA synthesis in each replicon is initiated at an origin of replication. In both budding yeast, Saccharomyces cerevisiae and fission yeast, Schizosaccharomyces pombe, origins contain specific sequences that are essential for initiation, although these differ significantly between the two yeasts with those of S. pombe being more complex then those of S. cerevisiae. However, it is not yet clear whether the replication origins of plants contain specific essential sequences or whether origin sites are determined by features of chromatin structure. In all eukaryotes there are several biochemical events that must take place before initiation can occur. These are the marking of the origins by the origin recognition complex (ORC), the loading onto the origins, in a series of steps, of origin activation factors including the MCM proteins, and the initial denaturation of the double helix to form a replication "bubble". Only then can the enzymes that actually initiate replication, primase and DNA polymerase-alpha, gain access to the template. In many cells this complex series of events occurs only once per cell cycle, ensuring that DNA is not re-replicated within one cycle. However, regulated re-replication of DNA within one cell cycle (DNA endoreduplication) is relatively common in plants, indicating that the "once-per-cycle" controls can be overridden.     

Analysis of soybean chloroplast DNA replication by two-dimensional gel electrophoresis

Chloroplast DNA replication was studied in the green, autotrophic suspension culture line SB-1 of Glycine max. Three regions (restriction fragments Sac I 14.5, Pvu II 4.1 and Pvu II 14.8) on the plastome were identified that displayed significantly higher template activity in in vitro DNA replication assays than all other cloned restriction fragments of the organelle genome, suggesting that these clones contain sequences that are able to direct initiation of DNA replication in vitro. In order to confirm that the potential in vitro origin sites are functional in vivo as well, replication intermediates were analyzed by two-dimensional gel electrophoresis using cloned restriction fragments as probes. The two Pvu II fragments that supported deoxynucleotide incorporation in vitro apparently do not contain a functional in vivo replication origin since replication intermediates from these areas of the plastome represent only fork structures. The Sac I 14.5 chloroplast DNA fragment, on the other hand, showed intermediates consistent with a replication bubble originating within its borders, which is indicative of an active in vivo origin. Closer examination of cloned Sac I 14.5 sub-fragments confirmed high template activity in vitro for two, S/B 5 and S/B 3, which also seem to contain origin sites utilized in vivo as determined by two-dimensional gel electrophoresis. The types of replication intermediate patterns obtained for these sub-fragments are consistent with the double D-loop model for chloroplast DNA replication with both origins being located in the large unique region of the plastome [17, 18]. This is the first report of a chloroplast DNA replication origin in higher plants that has been directly tested for in vivo function.     

Genomic mapping of single-stranded DNA in hydroxyurea-challenged yeasts identifies origins of replication

During DNA replication one or both strands transiently become single stranded: first at the sites where initiation of DNA synthesis occurs (known as origins of replication) and subsequently on the lagging strands of replication forks as discontinuous Okazaki fragments are generated. We report a genome-wide analysis of single-stranded DNA (ssDNA) formation in the presence of hydroxyurea during DNA replication in wild-type and checkpoint-deficient rad53 Saccharomyces cerevisiae cells. In wild-type cells, ssDNA was first observed at a subset of replication origins and later 'migrated' bi-directionally, suggesting that ssDNA formation is associated with continuously moving replication forks. In rad53 cells, ssDNA was observed at virtually every known origin, but remained there over time, suggesting that replication forks stall. Telomeric regions seemed to be particularly sensitive to the loss of Rad53 checkpoint function. Replication origins in Schizosaccharomyces pombe were also mapped using our method.     

Identification of a predominant replication origin in fission yeast.

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.     

Mapping an initiation region of DNA replication at a single-copy chromosomal locus in Drosophila melanogaster cells by two-dimensional gel methods and PCR-mediated nascent-strand analysis: multiple replication origins in a broad zone.

We have mapped an initiation region of DNA replication at a single-copy chromosomal locus in exponentially proliferating Drosophila tissue culture cells, using two-dimensional (2D) gel replicon mapping methods and PCR-mediated analysis of nascent strands. The initiation region was first localized downstream of the DNA polymerase alpha gene by determining direction of replication forks with the neutral/alkaline 2D gel method. Distribution of replication origins in the initiation region was further analyzed by using two types of 2D gel methods (neutral/neutral and neutral/alkaline) and PCR-mediated nascent-strand analysis. Results obtained by three independent methods were essentially consistent with each other and indicated that multiple replication origins are distributed in a broad zone of approximately 10 kb. The nucleotide sequence of an approximately 20-kb region that encompasses the initiation region was determined and searched for sequence elements potentially related to function of replication origins.     

Electron microscopic localization of replication origins in Oenothera chloroplast DNA

Summary The origins of chloroplast DNA (cpDNA) replication were mapped in two plastome types of Oenothera in order to determine whether variation in the origin of cpDNA replication could account for the different transmission abilities associated with these plastomes. Two pairs of displacement loop (D-loop) initiation sites were observed on closed circular cpDNA molecules by electron microscopy. Each pair of D-loops was mapped to the inverted repeats of the Oenothera cpDNA by the analysis of restriction fragments. The starting points of the two adjacent D-loops are approximately 4 kb apart, bracketing the 16S rRNA gene. Although there are small DNA length variations near one of the D-loop initiation sites, no apparent differences in the number and the location of replication origins were observed between plastomes with the highest (type I) and lowest (type IV) transmission efficiencies.     

Site-Specific DNA Binding of the Schizosaccharomyces pombe Origin Recognition Complex Is Determined by the Orc4 Subunit

The mechanism by which origin recognition complexes (ORCs) identify replication origins was investigated using purified Orc proteins from Schizosaccharomyces pombe. Orc4p alone bound tightly and specifically to several sites within S. pombe replication origins that are genetically required for origin activity. These sites consisted of clusters of A or T residues on one strand but were devoid of either alternating A and T residues or GC-rich sequences. Addition of a complex consisting of Orc1, -2, -3, -5, and -6 proteins (ORC-5) altered neither Orc4p binding to origin DNA nor Orc4p protection of specific sequences. ORC-5 alone bound weakly and nonspecifically to DNA; strong binding required the presence of Orc4p. Under these conditions, all six subunits remained bound to chromatin isolated from each phase of the cell division cycle. These results reveal that the S. pombe ORC binds to multiple, specific sites within replication origins and that site selection, at least in vitro, is determined solely by the Orc4p subunit.     

Short DNA fragments without sequence similarity are initiation sites for replication in the chromosome of the yeast Yarrowia lipolytica

We have previously shown that both a centromere (CEN) and a replication origin are necessary for plasmid maintenance in the yeast Yarrowia lipolytica (). Because of this requirement, only a small number of centromere-proximal replication origins have been isolated from Yarrowia. We used a CEN-based plasmid to obtain noncentromeric origins, and several new fragments, some unique and some repetitive sequences, were isolated. Some of them were analyzed by two-dimensional gel electrophoresis and correspond to actual sites of initiation (ORI) on the chromosome. We observed that a 125-bp fragment is sufficient for a functional ORI on plasmid, and that chromosomal origins moved to ectopic sites on the chromosome continue to act as initiation sites. These Yarrowia origins share an 8-bp motif, which is not essential for origin function on plasmids. The Yarrowia origins do not display any obvious common structural features, like bent DNA or DNA unwinding elements, generally present at or near eukaryotic replication origins. Y. lipolytica origins thus share features of those in the unicellular Saccharomyces cerevisiae and in multicellular eukaryotes: they are discrete and short genetic elements without sequence similarity.     

Determination of human DNA replication origin position and efficiency reveals principles of initiation zone organisation

Replication of the human genome initiates within broad zones of ∼150 kb. The extent to which firing of individual DNA replication origins within initiation zones is spatially stochastic or localised at defined sites remains a matter of debate. A thorough characterisation of the dynamic activation of origins within initiation zones is hampered by the lack of a high-resolution map of both their position and efficiency. To address this shortcoming, we describe a modification of initiation site sequencing (ini-seq), based on density substitution. Newly replicated DNA is rendered ‘heavy-light’ (HL) by incorporation of BrdUTP while unreplicated DNA remains ‘light-light’ (LL). Replicated HL-DNA is separated from unreplicated LL-DNA by equilibrium density gradient centrifugation, then both fractions are subjected to massive parallel sequencing. This allows precise mapping of 23,905 replication origins simultaneously with an assignment of a replication initiation efficiency score to each. We show that origin firing within early initiation zones is not randomly distributed. Rather, origins are arranged hierarchically with a set of very highly efficient origins marking zone boundaries. We propose that these origins explain much of the early firing activity arising within initiation zones, helping to unify the concept of replication initiation zones with the identification of discrete replication origin sites.     

Purification and characterization of the Schizosaccharomyces pombe origin recognition complex: interaction with origin DNA and Cdc18 protein

The origin recognition complex (ORC) plays a central role in the initiation of DNA replication in eukaryotic cells. It interacts with origins of DNA replication in chromosomal DNA and recruits additional replication proteins to form functional initiation complexes. These processes have not been well characterized at the biochemical level except in the case of Saccharomyces cerevisiae ORC. We report here the expression, purification, and initial characterization of Schizosaccharomyces pombe ORC (SpORC) containing six recombinant subunits. Purified SpORC binds efficiently to the ars1 origin of DNA replication via the essential Nterminal domain of the SpOrc4 subunit which contains nine AT-hook motifs. Competition binding experiments demonstrated that SpORC binds preferentially to DNA molecules rich in AT-tracts, but does not otherwise exhibit a high degree of sequence specificity. The complex is capable of binding to multiple sites within the ars1 origin of DNA replication with similar affinities, indicating that the sequence requirements for origin recognition in S. pombe are significantly less stringent than in S. cerevisiae. We have also demonstrated that SpORC interacts directly with Cdc18p, an essential fission yeast initiation protein, and recruits it to the ars1 origin in vitro. Recruitment of Cdc18p to chromosomal origins is a likely early step in the initiation of DNA replication in vivo. These data indicate that the purified recombinant SpORC retains at least two of its primary biological functions and that it will be useful for the eventual reconstitution of the initiation reaction with purified proteins.     

Functional mapping and DNA sequence of an equine herpesvirus 1 origin of replication.

The genome of equine herpesvirus 1 (EHV-1) defective interfering (DI) particle DNA originates from discrete regions within the standard (STD) EHV-1 genome: the left terminus (0.0 to 0.04 map units) and the inverted repeats (0.78 to 0.79 and 0.83 to 0.87 map units of the internal inverted repeat; 0.91 to 0.95 and 0.99 to 1.00 map units of the terminal inverted repeat). Since DI DNA must contain cis-acting DNA sequences, such as replication origins, which cannot be supplied in trans by the STD EHV-1 virus, regions of the EHV-1 genome shown to be in DI DNA were assayed for the presence of a viral origin of DNA replication. Specifically, STD EHV-1 DNA fragments encompassing the genomic regions present in DI particle DNA were inserted into the vector pAT153, and individual clones were tested by transfection assays for the ability to support the amplification and replication of plasmid DNA in EHV-1-infected cells. The Sma-1 subfragment of the internal inverted repeat sequence (0.83 to 0.85 map units) was shown to contain origin of replication activity. Subcloning and BAL 31 deletion analysis of the 2.35-kilobase-pair (kbp) Sma-1 fragment delineated a 200-bp fragment that contained origin activity. The origin activities of all EHV-1 clones which were positive by the transfection assay were confirmed by methylation analysis by using the methylation-sensitive restriction enzymes DpnI and MboI. DNA sequencing of the 200-bp fragment which contained an EHV-1 origin of replication indicated that this region has significant homology to previously characterized origins of replication of human herpesviruses. Furthermore, comparison of known origin sequences demonstrated that a 9-bp sequence, CGTTCGCAC, which is conserved among all origins of replication of human lytic herpesviruses and which is contained within the 18-bp region in herpes simplex virus type 1 origins shown by others to be protected by an origin-binding protein (P. Elias, M. E. O'Donnell, E. S. Mocarski, and I. R. Lehman, Proc. Natl. Acad. Sci. USA 83:6322-6326) is also conserved across species in the EHV-1 origin of replication.     

Genome-wide characterization of fission yeast DNA replication origins

Eukaryotic DNA replication is initiated from multiple origins of replication, but little is known about the global regulation of origins throughout the genome or in different types of cell cycles. Here, we identify 401 strong origins and 503 putative weaker origins spaced in total every 14 kb throughout the genome of the fission yeast Schizosaccharomyces pombe. The same origins are used during premeiotic and mitotic S-phases. We found that few origins fire late in mitotic S-phase and that activating the Rad3 dependent S-phase checkpoint by inhibiting DNA replication had little effect on which origins were fired. A genome-wide analysis of eukaryotic origin efficiencies showed that efficiency was variable, with large chromosomal domains enriched for efficient or inefficient origins. Average efficiency is twice as high during mitosis compared with meiosis, which can account for their different S-phase lengths. We conclude that there is a continuum of origin efficiency and that there is differential origin activity in the mitotic and meiotic cell cycles.     

Evidence that replication initiates at only some of the potential origins in each oligomeric form of bovine papillomavirus type 1 DNA.

In a subclone of ID13 mouse fibroblasts latently infected with bovine papillomavirus type 1 (BPV-1) DNA, the viral genome occurred as a mixture of extrachromosomal circular monomers and oligomers. Multiple copies were also associated with the host cell genome, predominantly at a single site in a head-to-tail tandem array. We examined the replicative intermediates of extrachromosomal forms of BPV-1 DNA by using two-dimensional gel electrophoresis. The results obtained indicate that initiation of DNA replication occurred near the center of the EcoRI-BamHI 5.6-kilobase fragment. In some molecules, however, this fragment was replicated from one end to the other by means of a single fork initiated elsewhere. Termination also occurred within this fragment. The EcoRI-BamHI 2.3-kilobase fragment replicated as a DNA molecule containing a termination site for DNA replication and also by means of a single fork traversing the fragment from one end to the other. Thus, replication forks proceeded through these fragments in different manners, apparently depending on whether they were part of a monomer, a dimer, a trimer, or higher oligomers. These observations lead to the conclusion that initiation of DNA replication in BPV-1 DNA takes place at or close to plasmid maintenance sequence 1. From this point, replication proceeds bidirectionally and termination occurs approximately 180 degrees opposite the origin. The results obtained are consistent with one or more replication origins being quiescent in BPV-1 DNA oligomers.     

Sequence-specific interactions between cellular DNA-binding proteins and the adenovirus origin of DNA replication.

The adenovirus origin of DNA replication contains three functionally distinct sequence domains (A, B, and C) that are essential for initiation of DNA synthesis. Previous studies have shown that domain B contains the recognition site for nuclear factor I (NF-I), a cellular protein that is required for optimal initiation. In the studies reported here, we used highly purified NF-I, prepared by DNA recognition site affinity chromatography (P. J. Rosenfeld and T. J. Kelly, Jr., J. Biol. Chem. 261:1398-1408, 1986), to investigate the cellular protein requirements for initiation of viral DNA replication. Our data demonstrate that while NF-I is essential for efficient initiation in vitro, other cellular factors are required as well. A fraction derived from HeLa cell nuclear extract (BR-FT fraction) was shown to contain all the additional cellular proteins required for the complete reconstitution of the initiation reaction. Analysis of this complementing fraction by a gel electrophoresis DNA-binding assay revealed the presence of two site-specific DNA-binding proteins, ORP-A and ORP-C, that recognized sequences in domains A and C, respectively, of the viral origin. Both proteins were purified by DNA recognition site affinity chromatography, and the boundaries of their binding sites were defined by DNase I footprint analysis. Additional characterization of the recognition sequences of ORP-A, NF-I, and ORP-C was accomplished by determining the affinity of the proteins for viral origins containing deletion and base substitution mutations. ORP-C recognized a sequence between nucleotides 41 and 51 of the adenovirus genome, and analysis of mutant origins indicated that efficient initiation of replication is dependent on the presence of a high-affinity ORP-C-binding site. The ORP-A recognition site was localized to the first 12 base pairs of the viral genome within the minimal origin of replication. These data provide evidence that the initiation of adenovirus DNA replication involves multiple protein-DNA interactions at the origin.