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PubMed1991

Multicomponent origin of cytomegalovirus lytic-phase DNA replication.

Cytomegalovirus (CMV) lytic-phase DNA replication requires both trans-acting factors, such as the virus-coded DNA polymerase, and a previously undefined cis-acting element, the origin, within which initiation occurs. We have located a candidate origin of CMV lytic-phase DNA replication, oriLyt, in both simian and human strains by assessing the ability of cloned restriction fragments to mediate phosphonoformic acid-sensitive DNA replication after transfection into human fibroblasts when required trans-acting factors were supplied by infection. In initial experiments the simian CMV-like strain Colburn EcoRI D fragment directed DNA replication; this fragment contains all of the single-stranded DNA-binding protein gene (dbp) and about 7 kbp of upstream sequence. A larger region upstream of human CMV dbp also mediated replication in transient assays. Subsequent subcloning and deletion analyses defined a CMV strain Colburn region sufficient for origin function, spanning about 1,300 bp in the apparently noncoding region upstream of dbp. The nucleotide sequence of this region revealed four distinct domains, containing (i) a 9-bp repeated sequence, (ii) an A+T-rich segment, (iii) an 11-bp direct repeat, and (iv) a 47-bp direct repeat. At least some part of each of these domains was required for origin function. Therefore, like the Epstein-Barr virus lytic-phase origin of DNA replication, CMV oriLyt appears to be structurally complex.

Base Sequence↗
PubMed2003

Sequence diversity and functional conservation of the origin of replication in lactococcal prolate phages.

Prolate or c2-like phages are a large homologous group of viruses that infect the bacterium Lactococcus lactis. In a collection of 122 prolate phages, three distinct, non-cross-hybridizing groups of origins of DNA replication were found. The nonconserved sequence was confined to the template for an untranslated transcript, P(E)1-T, 300 to 400 nucleotides in length, while the flanking sequences were conserved. All three origin types, despite the low sequence homology, have the same functional characteristics: they express abundant P(E)1-T transcripts and can function as origins of plasmid replication in the absence of phage proteins. Using chimeric constructs, we showed that hybrids of two nonhomologous origin sequences failed to function as replication origins, suggesting that preservation of a particular secondary structure of the P(E)1-T transcript is required for replication. This is the first systematic survey of the sequence and function of origins of replication in a group of lactococcal phages.

Bacteriophages↗
PubMed1974

Origin and direction of Haemophilus bacteriophage HP1 DNA replication.

Rapidly growing Haemophilus influenzae strain Rd bacteria were infected with bacteriophage HP1 and DNA extracts prepared at various times thereafter. A number of phage genes scattered along the entire phage genome were quantitatively assayed by transformation. The kinetics of activity increases of these genes suggests that phage HP1 DNA replication begins at a fixed origin about one-quarter from the right end and that it proceeds to the left.

Bacteriophages↗
PubMed1998

Origin DNA-binding proteins.

The first step in DNA replication involves the recognition of origin DNA sequences by origin-binding proteins. The three-dimensional structures of three different origin DNA-binding proteins have recently been solved. These proteins form a structural class distinct from other DNA-binding proteins. One of the origin-binding proteins, Epstein-Barr virus nuclear antigen 1, most likely has two modes of DNA binding; the sequential use of these modes may be important for the initiation of DNA replication.

Antigens, Polyomavirus Transforming↗
PubMed1980

Replication origin of the Escherichia coli K-12 chromosome: the size and structure of the minimum DNA segment carrying the information for autonomous replication.

A DNA fragment containing the replication origin of the Eschericia coli K-12 chromosome was inserted in two correlations at either the BamHI or SalI site of pBR322 DNA. All the resulting hybrid plasmids were found to replicate in both polA and polA+ cells, whereas pBR322 replicates only in polA+ cells. This characteristic provided a method for assaying the autonomously replicating ability (Ori function of the E. coli origin. In order to define the minimum DNA region (ori) that determines Ori function, deletions of various sizes were introduced from either side of the ori-containing segment in the hybrid plasmids by in vitro techniques, and the correlation between the Ori phenotype and nucleotide sequence of the deletion derivatives was analyzed. It was found that the left end of ori is between positions 23 and 35, and the right end is either position 266 or 267 in our nucleotide coordinate (Sugimoto et al., 1979). Therefore, ori is present within a region of minimum 232 base pairs and maximum 245 base pairs in length. The Ori+ and Ori- phenotypes were clearly resolved at both sides of these boundaries by the above assay procedure. To obtain information about the effect of mutations in the internal region of the defined ori stretch, short sequences were inserted or deleted in vitro in the vicinity of several restriction sites within ori on the hybrid plasmids. Most of these plasmids carrying modified sequences showed Ori- phenotype, suggesting that most parts of the ori stretch play important roles in Ori function.

Base Sequence↗
PubMed1998

oriGNAI3: a narrow zone of preferential replication initiation in mammalian cells identified by 2D gel and competitive PCR replicon mapping techniques.

The nature of mammalian origins of DNA replication remains controversial and this is primarily because two-dimensional gel replicon mapping techniques have identified broad zones of replication initiation whereas several other techniques, such as quantitative PCR, have disclosed more discrete sites of initiation at the same chromosomal loci. In this report we analyze the replication of an amplified genomic region encompassing the 3'-end of the GNAI3 gene, the entire GNAT2 gene and the intergenic region between them in exponentially growing Chinese hamster fibroblasts. These cells express GNAI3 but not GNAT2 . The replication pattern was first analyzed by two-dimensional neutral-alkaline gel electrophoresis. Surprisingly, the results revealed a small preferential zone of replication initiation, of at most 1.7 kb, located in a limited part of the GNAI3 - GNAT2 intergenic region. Mapping of this initiation zone was then confirmed by quantitative PCR. The agreement between the two techniques exploited here strengthens the hypothesis that preferred sites of replication initiation do exist in mammalian genomes.

AMP Deaminase↗
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↗
PubMed2000

Analyzing DNA strand compositional asymmetry to identify candidate replication origins of Borrelia burgdorferi linear and circular plasmids.

The Lyme disease agent Borrelia burgdorferi has a genome composed of a linear chromosome and a series of linear and circular plasmids. We previously mapped the oriC of the linear chromosome to the center of the molecule, where a pronounced switch in CG skew occurs. In this study, we analyzed B. burgdorferi plasmid sequences for AT and CG skew in an effort to similarly identify plasmid replication origins. Cumulative skew diagrams of the plasmids suggested that they, like the linear chromosome, replicate bidirectionally from an internal origin. The B. burgdorferi linear chromosome contains homologs to partitioning protein genes soj and spoOJ, which are closely linked to oriC at the minimum cumulative skew point of the 1-Mb molecule. A soj/parA homolog also maps to cumulative skew minima of the B. burgdorferi linear and circular plasmids, further suggesting that these regions contain the replication origin. The heterogeneity in these genes and in the nucleotide sequences of the putative origin regions could account for the mutual compatibility of the multiple DNA elements in B. burgdorferi.

Base Composition↗
PubMed2002

Separation of the DNA replication, segregation, and transcriptional activation functions of Epstein-Barr nuclear antigen 1.

In latent Epstein-Barr virus infection, the viral EBNA1 protein binds to specific sites in the viral origin of DNA replication, oriP, to activate the initiation of DNA replication, enhance the expression of other viral latency proteins, and partition the viral episomes during cell division. The DNA binding domain of EBNA1 is required for all three function, and a Gly-Arg-rich sequence between amino acids 325 and 376 is required for both the transcriptional activation and partitioning functions. We have used mutational analysis to identify additional EBNA1 sequences that contribute to EBNA1 functions. We show that EBNA1 amino acids 8 to 67 contribute to, but are not absolutely required for, EBNA1 replication, partitioning, and transcriptional activation functions. A Gly-Arg-rich sequence (amino acids 33 to 53) that is similar to that of amino acids 325 to 376 and lies within the 8-to-67 region was not responsible for the functional contributions of residues 8 to 67, since deletion of amino acids 34 to 52 alone did not affect EBNA1 functions. We also found that deletion of amino acids 61 to 83 eliminated the transcriptional activity of EBNA1 without affecting partitioning. This mutant also exhibited an increased replication efficiency that resulted in the maintenance of oriP plasmids at a copy number approximately fourfold higher than for wild-type EBNA1. The results indicate that the three EBNA1 functions have overlapping but different sequence requirements. Transcriptional activation requires residues 61 to 83 and 325 to 376 and is stimulated by residues 8 to 67; partitioning requires residues 325 to 376 and is stimulated by residues 8 to 67; and replication involves redundant contributions of both the 325-to-376 and 8-to-67 regions.

Cell Line↗
PubMed1999

Mechanism of DNA replication in eukaryotic cells: cellular host factors stimulating adenovirus DNA replication.

Replication of adenovirus (Ad) DNA depends on interactions between three viral and three cellular proteins. Human transcription factors NFI and Oct-1 recruit the Ad DNA polymerase to the origin of DNA replication as a complex with the Ad protein primer pTP. High affinity and specificity DNA binding to recognition sites in this origin by the transcription factors stimulate and stabilize pre-initiation complex formation to compensate for the low binding specificity of the pTP/pol complex. In this review, we discuss the properties of NFI and Oct-1 and the mechanism by which they enhance initiation of DNA replication. We propose a model that describes the dynamics of initiation and elongation as well as the assembly and disassembly of the pre-initiation complex.

Adenoviridae↗
PubMed1983

Genomic structure of human polyoma virus JC: nucleotide sequence of the region containing replication origin and small-T-antigen gene.

The nucleotide sequence of the region of human polyoma virus JC DNA between 0.5 and 0.7 map units from a unique EcoRI cleavage site was determined and compared with those of the corresponding regions of another human polyoma virus, BK, and simian virus 40 DNAs. Within this region consisting of 945 base pairs, we located the origin of DNA replication near 0.7 map units, the entire coding region for small T antigen, and the splice junctions for large-T-antigen mRNA. The deduced amino acid sequences for small T antigen and the part of large T antigen markedly resembled those of polyoma virus BK and simian virus 40. The results strongly suggest that polyoma virus JC has the same organization of early genome as polyoma virus BK and simian virus 40 on the physical map, with the EcoRI site as a reference point.

Amino Acid Sequence↗
PubMed1986

M13-oriC cloning vehicles: use for amplification of high copy lethal (HCL) genetic elements.

M13 cloning vehicles have been constructed which contain the Escherichia coli origin for DNA replication (oriC), with and without selectable antibiotic-resistance genes. Since the M13 viral strand origin requires a functional rep gene product, using oriC these vehicles propagate as low-copy-number plasmids in E. coli rep mutants. This property is exploited to amplify cloned "high copy lethal" (HCL) DNA fragments, those containing genetic elements which kill the E. coli host when present at multiple copies in the cell. Following cloning of such fragments in these vehicles and initial selection in E. coli rep cells, the M13-oriC chimeric plasmid DNA is used to transfect appropriate E. coli rep+ cells. The chimeric DNA propagates as M13 viral DNA, yielding double-stranded and single-stranded DNA products and phage particles prior to killing of the host via expression of the HCL element; these events mimic a lytic phage infection. Such amplification will greatly facilitate both DNA "library" constructions (HCL elements are absent a priori from libraries using high-copy-number cloning vehicles) and studies of HCL elements including restriction mapping, DNA sequencing, and physiological studies.

Chromosome Mapping↗
PubMed1999

C8-guanine adduct-induced stabilization of a -1 frame shift intermediate in a nonrepetitive DNA sequence.

The mechanism of frame shift mutagenesis induced by N-(deoxyguanosin-8-yl)-1-aminopyrene, the major DNA adduct formed by the carcinogen 1-nitropyrene, was investigated by thermal melting studies of a 13-mer in which the adduct was flanked by a 5' and a 3' C. Compared to the unmodified 13-mer, the adduct destabilized the duplex by 4-5 kcal/mol, and the DeltaDeltaG value remained approximately the same regardless of which base was placed opposite the adduct. In contrast, deletion of the base opposite the adduct stabilized the duplex by nearly 4 kcal/mol. The adduct in the same sequence context was inserted into a bacteriophage M13 DNA containing the simian virus 40 origin of replication. The constructed DNA template was replicated in vitro with extracts from normal human fibroblasts. The adduct was not removed from the progeny DNA following bidirectional semiconservative replication, which suggests that it had been bypassed, rather than repaired, by the cell extract. When newly replicated bacteriophage was evaluated for mutations in the region of the modified G, most contained a G at the adduct site, indicating error-free replication. A small number of mutants ( approximately 2 x 10(-3)) were detected, all of which contained a targeted G.C base pair deletion. This suggests a relationship between the thermodynamic stability of the adduct in DNA and the errors that occurred during replicative bypass by the human DNA polymerases.

Bacteriophage M13↗
PubMed1994

Novel replication inhibitory function of the developmental regulator/transcription repressor protein WT1 encoded by the Wilms' tumor gene.

The tumor suppressor/developmental regulator protein WT1 encoded by the Wilms' tumor gene is a zinc finger-containing transcription factor which binds to the G+C-rich motif 5'-GCGGGGGCG-3' and represses transcription. Alternatively spliced variants of WT1 (termed+KTS) having an insertion in the zinc finger region are defective for binding to and hence for repression of transcription from promoters containing this motif. Due to the known interactions of two other tumor suppressor proteins with the simian virus 40 (SV40) oncoprotein large tumor antigen (TAg) [which in one case (p53) results in inhibition of the replication initiation activity of TAg], and because of the presence of G+C-rich sequences in the SV40 origin region, we tested the effect of WT1 on TAg- and SV40 origin-dependent DNA replication. WT1 and its alternatively spliced variants were found to be potent inhibitors of replication. Inhibition of replication by WT1 required portions of the N-terminal transcription repression domain and the C-terminal DNA binding domain, while other WT1 sequences needed for transcriptional regulation were dispensable. WT1 neither inhibited the synthesis of TAg nor formed a stable complex with it. Studies of the requirement of cis-active origin sequences in vivo and protein-DNA interactions in vitro indicated that WT1 and its alternatively spliced variants might inhibit replication by their novel binding to the GC box promoter motifs of the SV40 21 bp repeat replication-auxiliary sequence.

Alternative Splicing↗
PubMed2000

Association of human origin recognition complex 1 with chromatin DNA and nuclease-resistant nuclear structures.

An origin recognition complex (ORC) consisting of six polypeptides has been identified as a DNA replication origin-binding factor in Saccharomyces cerevisiae. Homologues of ORC subunits have been discovered among eukaryotes, and we have prepared monoclonal antibodies against a human homologue of ORC1 (hORC1) to study its localization in human cells. It was thus found to associate with nuclei throughout the cell cycle and to be resistant to nonionic detergent treatment, in contrast to MCM proteins, which are other replication factors, the association of which with nuclei is clearly dependent on the phase of the cell cycle. A characteristic feature of hORC1 is dissociation by NaCl in a narrow concentration range around 0.25 M, suggesting interaction with some specific partner(s) in nuclei. Nuclease treatment experiments and UV cross-linking experiments further indicated interaction with both nuclease-resistant nuclear structures and chromatin DNA. Although its DNA binding was unaffected, some variation in the cell cycle was apparent, the association with nuclear structures being less stable in the M phase. Interestingly, the less stable association occurred concomitantly with hyperphosphorylation of hORC1, suggesting that this hyperphosphorylation may be involved in M phase changes.

Antibodies, Monoclonal↗
PubMed1996

Insufficient levels of NFIII and its low affinity for the origin of adenovirus type 12 (Ad12) DNA replication contribute to the abortive infection of BHK21 hamster cells by Ad12.

Human adenovirus type 12 (Ad12) induces undifferentiated sarcomas in neonate Syrian hamsters and hence presents a suitable model for studies of the molecular mechanism of viral oncogenesis. Since we submit that an understanding of the early steps in the interaction between Ad12 and hamster cells might shed light on the initiation of malignant transformation, the abortive infection of BHK21 hamster cells with Ad12 has been investigated in detail. Ad12 replication in these cells is blocked in early stages, while Ad2 can replicate to moderate titers. Early Ad12 genes are expressed in BHK21 hamster cells, but there is a total block in Ad12 DNA replication and late gene transcription. The Ad5-transformed hamster cell line BHK297-C131, with the left terminus of Ad5 DNA chromosomally integrated and constitutively expressed, allows limited levels of Ad12 DNA replication and late transcription, probably through Ad5 E1 functions, but not the translation of late Ad12 gene products. We have now investigated the capacities of binding of nuclear proteins NFI and NFIII from permissive human KB cells, nonpermissive hamster BHK21 cells, and complementing BHK297-C131 cells to the origin of replication (ori) of Ad2 or Ad12 DNA. The electrophoretic mobility shift assay has been used to assess these binding reactions. The data support the notions that NFIII of BHK21 cells has a lower affinity for the ori of Ad12 DNA than for the ori of Ad2 DNA and that the levels of NFIII in BHK21 cells are markedly reduced compared with the levels in the permissive human KB cells or the complementing BHK297-C131 hamster cells. These deficiencies are contributing factors for the abortive infection of BHK21 hamster cells with Ad12. The lack of sufficient levels of NFIII in BHK21 cells is also consistent with the decreased replication capacity of Ad2 in hamster compared with human cell lines.

Adenoviruses, Human↗
PubMed1994

Analysis of an origin of DNA amplification in Sciara coprophila by a novel three-dimensional gel method.

The replication origin region for DNA amplification in Sciara coprophila DNA puff II/9A was analyzed with a novel three-dimensional (3D) gel method. Our 3D gel method involves running a neutral/neutral 2D gel and then cutting out vertical gel slices from the area containing replication intermediates, rotating these slices 90 degrees to form the third dimension, and running an alkaline gel for each of the slices. Therefore, replication intermediates are separated into forks and bubbles and then are resolved into parental and nascent strands. We used this technique to determine the size of forks and bubbles and to confirm the location of the major initiation region previously mapped by 2D gels to a 1-kb region. Furthermore, our 3D gel analyses suggest that only one initiation event in the origin region occurs on a single DNA molecule and that the fork arc in the composite fork-plus-bubble pattern in neutral/neutral 2D gels does not result from broken bubbles.

Animals↗