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PubMed1984

Structure and function of the adenovirus origin of replication.

Efficient initiation of adenovirus DNA replication requires the presence of specific terminal nucleotide sequences that collectively constitute the viral origin of replication. Using plasmids with deletions or base substitutions in a cloned segment of DNA derived from the terminus of the adenovirus 2 genome, we have demonstrated that the origin contains two functionally distinct regions. The first 18 bp of the viral genome are sufficient to support a limited degree of initiation. However, the presence of a sequence in the region between nucleotides 19 and 67 greatly enhances the efficiency of the initiation reaction. This region contains a specific binding site for a protein present in uninfected cells (KD = 2 X 10(-11) M). The bound protein protects the DNA segment between base pairs 19 and 43 from attack by DNAase I. Studies with deletion mutants indicate that binding of the cellular protein is responsible for the enhancement of initiation.

Adenoviruses, Human↗
PubMed1985

In vitro RNA polymerase interaction with a restriction fragment containing the Escherichia coli origin of replication.

The interaction of RNA polymerase with a restriction fragment containing the origin of Escherichia coli replication (oriC) was examined by methods used to investigate transcription promoter activities. Interactions of RNA polymerase with oriC were determined and characterized by agarose gel exclusion under conditions of polymerase binding and RNA synthesis initiation. These interactions were further demonstrated and defined by nitrocellulose retention experiments under various reaction conditions. The binding of RNA polymerase to the oriC fragment was compared to binding to the tetracycline promoter (tet), a known strong promoter of transcription. Specific localization of the RNA polymerase-oriC interaction was determined by restriction protection experiments. The binding of RNA polymerase was determined to be located near the HindIII site of oriC. These methods allowed the observation and characterization of a specific association of RNA polymerase with the origin of E. coli DNA replication.

DNA Replication↗
PubMed1988

ATP stimulates the binding of simian virus 40 (SV40) large tumor antigen to the SV40 origin of replication.

Simian virus 40 (SV40) large tumor antigen (T antigen) binds to two contiguous sites at the SV40 origin of replication. Of these two sites, I and II, only site II is critical for replication. We have studied the interaction between T antigen and these sites by two methods--nitrocellulose filter binding and DNase I protection. We show that T antigen binds with high occupancy to site I at 0 degrees C, 25 degrees C, and 37 degrees C but to site II only at 0 degrees C and 25 degrees C. At 37 degrees C, the temperature essential for the initiation of SV40 DNA replication in vitro, ATP is required for the interaction of T antigen and site II. ATP can be replaced efficiently by adenosine 5'-[beta,gamma-imido]triphosphate and ADP, suggesting that hydrolysis of the nucleotide is not essential for the binding of T antigen to site II. The binding to the region critical for replication can occur in the presence of a variety of nucleoside triphosphates; dATP supports binding at a concentration 1/30th that of ATP, while dGTP and rGTP were inactive at all concentrations tested.

Adenine Nucleotides↗
PubMed1992

Mode of replication, size and distribution of naturally occurring plasmids in Bacillus thuringiensis.

Cloned replication origin regions, derived from both small (4.9-7.5 MDa) and large (43-60 MDa) plasmids of Bacillus thuringiensis subspecies kurstaki strains HD73 and HD263 were used as hybridization probes in a Southern-blot analysis to assess both the size and horizontal distribution of native plasmid replicon groups among different subspecies of B. thuringiensis. In general, resident plasmids hybridizing to the replication origin regions derived from strains HD263 and HD73 were more commonly found in kurstaki strains than in non-kurstaki strains, suggesting a non-random distribution of plasmid incompatibility groups. Replication origin regions derived from the large HD263 plasmids (43-60 MDa) hybridized almost exclusively with large plasmids (greater than 30 MDa) of widely varying sizes. In contrast, replication origin regions derived from small plasmids hybridized exclusively with small plasmids (less than 10 MDa) showing little size variation. These results are consistent with previous observations concerning the relationship between plasmid size, mode of replication, and structural stability.

Bacillus thuringiensis↗
PubMed2003

Plasmid accumulation reduces life span in Saccharomyces cerevisiae.

Aging in the yeast Saccharomyces cerevisiae is under the control of multiple pathways. The production and accumulation of extrachromosomal rDNA circles (ERCs) is one pathway that has been proposed to bring about aging in yeast. To test this proposal, we have developed a plasmid-based model system to study the role of DNA episomes in reduction of yeast life span. Recombinant plasmids containing different replication origins, cis-acting partitioning elements, and selectable marker genes were constructed and analyzed for their effects on yeast replicative life span. Plasmids containing the ARS1 replication origin reduce life span to the greatest extent of the plasmids analyzed. This reduction in life span is partially suppressed by a CEN4 centromeric element on ARS1 plasmids. Plasmids containing a replication origin from the endogenous yeast 2 mu circle also reduce life span, but to a lesser extent than ARS1 plasmids. Consistent with this, ARS1 and 2 mu origin plasmids accumulate in approximately 7-generation-old cells, but ARS1/CEN4 plasmids do not. Importantly, ARS1 plasmids accumulate to higher levels in old cells than 2 mu origin plasmids, suggesting a correlation between plasmid accumulation and life span reduction. Reduction in life span is neither an indirect effect of increased ERC levels nor the result of stochastic cessation of growth. The presence of a fully functional 9.1-kb rDNA repeat on plasmids is not required for, and does not augment, reduction in life span. These findings support the view that accumulation of DNA episomes, including episomes such as ERCs, cause cell senescence in yeast.

Gene Dosage↗
PubMed1997

Origin-specific initiation of mammalian nuclear DNA replication in a Xenopus cell-free system.

The introduction of Chinese hamster ovary (CHO) cell nuclei into Xenopus egg extracts provides the only cell-free system that can efficiently initiate replication at a specific metazoan replication origin. With intact late-G1-phase nuclei as a substrate, the pattern of initiation sites for replication at the CHO dihydrofolate reductase (DHFR) locus is indistinguishable from that observed in cultured cells. By contrast, with early-G1-phase nuclei or with late-G1-phase nuclei that have damaged nuclear envelopes, these same extracts efficiently initiate replication at apparently random sites. Thus, at a distinct point during G1 phase [origin decision point (ODP)], nuclei experience a transition that is required for specific recognition of the DHFR origin by Xenopus egg cytosol. Described here are the basic requirements to achieve origin-specific initiation, which include: 1) a cell line that can be synchronized in G1 phase, 2) a method to prepare intact nuclei, 3) a technique to map origins with a few million cells, and 4) a small colony of Xenopus laevis. Immunodepletion of specific gene products allows one to test hypotheses about the requirements for origin recognition. Here we show that depletion of the Xenopus origin recognition complex subunit XORC2 from Xenopus egg extracts has no influence on the efficiency of replication or the pattern of initiation sites with either pre-ODP or post-ODP nuclei.

Animals↗
PubMed1999

E2F mediates developmental and cell cycle regulation of ORC1 in Drosophila.

Throughout the cell cycle of Saccharomyces cerevisiae, the level of origin recognition complex (ORC) is constant and ORCs are bound constitutively to replication origins. Replication is regulated by the recruitment of additional factors such as CDC6. ORC components are widely conserved, and it generally has been assumed that they are also stable factors bound to origins throughout the cell cycle. In this report, we show that the level of the ORC1 subunit changes dramatically throughout Drosophila development. The accumulation of ORC1 is regulated by E2F-dependent transcription. In embryos, ORC1 accumulates preferentially in proliferating cells. In the eye imaginal disc, ORC1 accumulation is cell cycle regulated, with high levels in late G1 and S phase. In the ovary, the sub-nuclear distribution of ORC1 shifts during a developmentally regulated switch from endoreplication of the entire genome to amplification of the chorion gene clusters. Furthermore, we find that overexpression of ORC1 alters the pattern of DNA synthesis in the eye disc and the ovary. Thus, replication origin activity appears to be governed in part by the level of ORC1 in Drosophila.

Animals↗
PubMed1989

Selective initiation of replication at origin sequences of the rDNA molecule of Physarum polycephalum using synchronous plasmodial extracts.

A cell-free system using synchronous plasmodial extracts initiates replication selectively on the 60 kb rDNA palindrome of Physarum polycephalum. Preferential labeling of rDNA fragments by nuclear extracts, in which elongation is limited, indicates that initiation occurs at two positions corresponding to in vivo origins of replication estimated by electron microscopy. Both nuclear and whole plasmodial extracts initiate selectively within a plasmid, pPHR21, containing one of these origins. In this plasmid bubbles expand bidirectionally and generate DpnI-resistant DNA. Extracts made at prophase or early S phase, times when the nucleolus is disorganized, are most active in pPHR21 replication. Mapping positions of replication bubbles locates the initiation point in a 3.2 kb BstEII fragment at the upstream border of a series of 31 bp repeats 2.4 kb from the initiation point for ribosomal gene transcription.

Base Sequence↗
PubMed1989

Analysis of unselected HSV-1 McKrae/HSV-2 HG 52 recombinants demonstrates preferential recombination between intact genomes and restriction endonuclease fragments containing an origin of replication.

To identify viral genes involved in reactivation of herpes simplex virus from latency, intertypic HSV-1 strain McKrae/HSV-2 strain HG 52 recombinants were selected following cotransfection of intact McKrae DNA and XbaI or HpaI cleaved HG 52 DNA. Eleven separately obtained recombinants containing HG 52 inserts between 0.35-0.56 and/or 0.82-1.0 map units (mu) were isolated. It was noted that with HpaI digested HG 52 DNA, only recombinants containing type 2 inserts from HpaI d (0.35-0.57) and/or containing an intact type 2 [S] region were isolated. Similarly with XbaI cleaved HG 52 DNA only recombinants containing type 2 sequences from XbaI c (0-0.45) were isolated. In effect, the type 2 insert always contained one or both origins of replication (ORIL/ORIS). In reciprocal experiments isolation of two recombinants from cotransfection of HpaI cleaved McKrae DNA with intact HG 52 DNA confirmed this finding; one contained both copies of ORIS and the intervening short region sequences of McKrae, the other contained approximately 3 kb of McKrae in which ORIL is located. These results indicate that either (a) the presence of an origin of replication in a RE fragment amplifies the fragment thereby increasing its concentration and hence recombination potential with intact genomes; and/or (b) recombination and replication may be correlated. In either case isolation of recombinants containing ORIL and ORIS from the restricted DNA parent strongly suggests that both origins are functional in vitro.

Crossing Over, Genetic↗
PubMed1999

The functional role of a DNA primase in chloroplast DNA replication in Chlamydomonas reinhardtii.

A complementation experiment was developed to identify the protein component that is essential for the in vitro replication of a cloned template containing a chloroplast DNA replication origin of Chlamydomonas reinhardtii. Using this method, we have identified a DNA primase activity that copurified with DNA polymerase from the crude protein mixture. The primase catalyzed the synthesis of short RNA primers on single-stranded DNA templates. Among the synthetic templates, the order of preference was poly(dA), poly(dT), and poly(dC). The primer size range for these templates was 11-18, 5-12, and 3-11 nucleotides, respectively. On a single-stranded template containing the chloroplast DNA replication origin, the primer length range reached 19 to 27 nucleotides, indicating a better processtivity. Several initiation sites were mapped on both strands of the cloned replication origin. Some preferential initiation sites were located on A tracks spaced at one helical turn apart within the bending locus. Primase improved the template specificity of the in vitro DNA replication system and enhanced the incorporation of radioactive dATP into the supercoiled template containing the core sequences of the chloroplast DNA replication origin.

Animals↗
PubMed1995

A single-stranded DNA binding protein binds the origin of replication of the duplex kinetoplast DNA.

Replication of the kinetoplast DNA (kDNA) minicircle of trypanosomatids initiates at a conserved 12-nt sequence, 5'-GGGGTTGGTGTA-3', termed the universal minicircle sequence (UMS). A sequence-specific single-stranded DNA-binding protein from Crithidia fasciculata binds the heavy strand of the 12-mer UMS. Whereas this UMS-binding protein (UMSBP) does not bind a duplex UMS dodecamer, it binds the double-stranded kDNA minicircle as well as a duplex minicircle fragment containing the origin-associated UMS. Binding of the minicircle origin region by the single-stranded DNA binding protein suggested the local unwinding of the DNA double helix at this site. Modification of thymine residues at this site by KMnO4 revealed that the UMS resides within an unwound or otherwise sharply distorted DNA at the minicircle origin region. Computer analysis predicts the sequence-directed curving of the minicircle origin region. Electrophoresis of a minicircle fragment containing the origin region in polyacrylamide gels revealed a significantly lower electrophoretic mobility than expected from its length. The fragment anomalous electrophoretic mobility is displayed only in its native conformation and is dependent on temperature and gel porosity, indicating the local curving of the DNA double helix. We suggest that binding of UMSBP at the minicircle origin of replication is possible through local unwinding of the DNA double helix at the UMS site. It is hypothesized here that this local melting is initiated through the untwisting of unstacked dinucleotide sequences at the bent origin site.

Animals↗
PubMed1992

Propagation of pSC101 plasmids defective in binding of integration host factor.

Integration host factor (IHF), a multifunctional protein of E. coli, normally is required for the replication of plasmid pSC101. T. T. Stenzel, P. Patel, and D. Bastia (Cell 49:709-717, 1987) have reported that IHF binds to a DNA locus near the pSC101 replication origin and enhances a static bend present in this region; mutation of the IHF binding site affects the plasmid's ability to replicate. We report here studies indicating that the requirement for IHF binding near the pSC101 replication origin is circumvented partially or completely by (i) mutation of the plasmid-encoded repA (replicase) gene or the chromosomally encoded topA gene, (ii) the presence on the plasmid of the pSC101 partition (par) locus, or (iii) replacement of the par locus by a strong transcriptional promoter. With the exception of the repA mutation, the factors that substitute for a functional origin region IHF binding site are known to alter plasmid topology by increasing negative DNA supercoiling, as does IHF itself. These results are consistent with the proposal that IHF binding near the pSC101 replication origin promotes plasmid replication by inducing a conformational change leading to formation of a repA-dependent DNA-protein complex. A variety of IHF-independent mechanisms can facilitate formation of the putative replication-initiation complex.

Bacterial Proteins↗
PubMed2003

Putative subunits of the maize origin of replication recognition complex ZmORC1-ZmORC5.

The finding in animal species of complexes homologous to the products of six Saccharomyces cerevisiae genes, origin of replication recognition complex (ORC), has suggested that ORC-related mechanisms have been conserved in all eukaryotes. In plants, however, the only cloned putative homologs of ORC subunits are the Arabidopsis ORC2 and the rice ORC1. Homologs of other subunits of plant origin have not been cloned and characterized. A striking observation was the absence from the Arabidopsis genome of an obvious candidate gene-homolog of ORC4. This fact raised compelling questions of whether plants, in general, and Arabidopsis, in particular, may have lost the ORC4 gene, whether ORC-homologous subunits function within a complex in plants, whether an ORC complex may form and function without an ORC4 subunit, whether a functional (but not sequence) protein homolog may have taken up the role of ORC4 in Arabidopsis, and whether lack of ORC4 is a plant feature, in general. Here, we report the first cloned and molecularly characterized five genes coding for the maize putative homologs of ORC subunits ZmORC1, ZmORC2, ZmORC3, ZmORC4 and ZmORC5. Their expression profiles in tissues with different cell-dividing activities are compatible with a role in DNA replication. Based on the potential of ORC-homologous maize proteins to bind each other in yeast, we propose a model for their possible assembly within a maize ORC. The isolation and molecular characterization of an ORC4-homologous gene from maize argues that, in its evolution, Arabidopsis may have lost the homologous ORC4 gene.

DNA Replication↗
PubMed1992

The E2 binding sites determine the efficiency of replication for the origin of human papillomavirus type 18.

Human papillomaviruses (HPV-s) have been shown to possess transforming and immortalizing activity for many different, mainly keratinocyte cell lines and they have been detected in 90% of anogenital cancer tissues, which suggests a causative role in the induction of anogenital and other tumours. We have exploited a quantitative assay to identify and characterize the origin of replication of the human papillomavirus type 18 (HPV-18), one of the most prevalent types in the high-risk HPV group. Replication of HPV origin fragments was studied transiently by cotransfection with a protein expression vector providing replication proteins E1 and E2. We have localized the HPV-18 origin to nucleotides 7767-119. This region contains three E2 binding sites and an essential A/T rich DNA region (nucleotides 9-35) that is partly homologous to the E1 binding site found in bovine papillomavirus type 1 (BPV-1) genome. At least one of the three E2 binding sites was absolutely required for origin function; addition of other E2 sites had cooperative stimulating effect. This is the first quantitative analysis of the E2 binding sites for papillomavirus replication.

Base Sequence↗
PubMed1988

Plant expression vectors with the origin of replication of the W-type plasmid Sa.

A new class of binary vectors has been constructed, containing the origin of replication of the W-type plasmid Sa. These vectors are designed to express foreign genes in plants under control of the TR gene 2' promoter or the promoter of a light-inducible ribulose-1,5-bisphosphate carboxylase small subunit gene of Arabidopsis thaliana.

Base Sequence↗
PubMed1994

Mispair-, site-, and strand-specific error rates during simian virus 40 origin-dependent replication in vitro with excess deoxythymidine triphosphate.

We have measured the fidelity of leading and lagging strand DNA replication in HeLa cell extracts. Providing an excess of one dNTP in reactions induces replication errors consistent with misincorporation of that dNTP. With excess dTTP, both substitutions and single-nucleotide frameshifts are induced. Error distribution is nonrandom; reproducible hot spots for a substitution and a frameshift error are observed. Measurements with two vectors having the origin of replication on opposite sides of the mutational target demonstrate that error rates for G.dTTP and C.dTTP mispairs depend on whether the strand is replicated as the leading or lagging strand. Also, the two hot spots are only observed in one origin-target orientation. Replication reactions reconstituted from two fractions derived from extracts are 3-fold less accurate, but the error specificity with excess dTTP is similar to that with extracts. This suggests that the processes responsible for the nonrandom error rates are not lost as a result of fractionation. Furthermore, the reconstituted system is devoid of mismatch repair activity. Thus, mismatch repair is not responsible for the mispair-, site-, and strand-specific differences observed.

Base Sequence↗
PubMed1989

Requirements for species-specific papovavirus DNA replication.

Replication of papovavirus DNA requires a functional replication origin, a virus-encoded protein, large T antigen, and species-specific permissive factors. How these components interact to initiate and sustain viral DNA replication is not known. Toward that end, we have attempted to identify the viral target(s) of permissive factors. The functionally defined replication origins of polyomavirus and simian virus 40, two papovaviruses that replicate in different species (mice and monkeys, respectively), are composed of two functionally distinct domains: a core domain and an auxiliary domain. The origin cores of the two viruses are remarkably similar in primary structure and have common binding sites for large T antigen. By contrast, their auxiliary domains share few sequences and serve as binding sites for cellular proteins. It seemed plausible, therefore, that if cellular permissive factors interacted with the replication origin, their targets were likely to be in the auxiliary domain. To test this hypothesis we constructed hybrid origins for DNA replication that were composed of the auxiliary domain of one virus and the origin core of the other and assessed their capacity to replicate in a number of mouse and monkey cell lines, which express the large T antigen of one or the other virus. The results of this analysis showed that the auxiliary domains of the viral replication origins could substitute for one another in DNA replication, provided that the viral origin core and its cognate large T antigen were present in a permissive cellular milieu. Surprisingly, the large T antigens of the viruses could not substitute for one another, regardless of the species of origin of the host cell, even though the two large T antigens bind to the same sequence motif in vitro. These results suggest that species-specific permissive factors do not interact with the origin-auxiliary domains but, rather, with either the origin core or the large T antigen or with both components to effect DNA replication.

Animals↗
PubMed1999

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.

Centromere↗