Sequence-specific DNA binding protein(s) that bind(s) to a putative human DNA replication origin.
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Cloning, Molecular↗
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Evidence suggesting that the ARS elements associated with silencers of the yeast mating-type locus HML do not function as chromosomal DNA replication origins.
The silent mating-type loci of Saccharomyces cerevisiae, HML and HMR, are flanked by transcriptional silencers that have ARS activity (i.e., they function as replication origins when in plasmids). To test whether these ARS elements are chromosomal origins, we mapped origins near HML (close to the left telomere of chromosome III). Our results indicate that the HML-associated ARS elements either do not function as chromosomal replication origins or do so at a frequency below our detection level, suggesting that replication from a silencer-associated origin in each S phase is not essential for the maintenance of transcriptional repression at HML. Our results also imply that the ability of a DNA fragment to function as an ARS element in a plasmid does not ensure its ability to function as an efficient chromosomal replication origin. Telomere proximity is not responsible for inactivating these ARS elements, because they are not detectably functional as chromosomal origins even in genetically modified strains in which they are far from the telomere.
Chromosome Mapping↗
Prokaryotic promoters in the chloroplast DNA replication origin of Chlamydomonas reinhardtii.
In Chlamydomonas reinhardtii, one displacement loop region which initiates the replication of chloroplast DNA was located on a 1.05 kb restriction fragment. This fragment was cloned and sequenced. In this report, the galK expression plasmid, pKO1 was used to screen for the presence of any prokaryotic promoter within the cloned fragment. The insertion of 2 AluI fragments yielded galK+ colonies. Sequence analyses of these AluI inserts revealed prokaryotic promoter consensus regions. Cloning into pKOTWI and subsequent DNA sequencing were used to determine the promoter-active orientation of each insert. Two back-to-back prokaryotic promoters were mapped on a 79 bp AluI fragment located within the displacement loop region.
Chlamydomonas↗
New beginnings in studies of eukaryotic DNA replication origins.
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Base Sequence↗
Evidence for the direct involvement of DNA replication origin in synthesis of late SV40 RNA.
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Animals↗
Transition in specification of embryonic metazoan DNA replication origins.
In early Xenopus embryos, in which ribosomal RNA genes (rDNA) are not transcribed, rDNA replication initiates and terminates at 9- to 12-kilobase pair intervals, with no detectable dependence on specific DNA sequences. Resumption of ribosomal RNA (rRNA) synthesis at late blastula and early gastrula is accompanied by a specific repression of replication initiation within transcription units; the frequency of initiation within intergenic spacers remains as high as in early blastula. These results demonstrate that for rRNA genes, circumscribed zones of replication initiation emerge in intergenic DNA during the time in metazoan development when the chromatin is remodeled to allow gene transcription.
Animals↗
Enzymatic recognition of DNA replication origins.
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Bacteria↗
Identification of critical elements within the JC virus DNA replication origin.
The T antigen of JC virus (JCV) does not interact productively with the simian virus 40 (SV40) origin of replication. In contrast, the SV40 T antigen does drive replication from the JCV origin as well as from its own. The basis for this restricted interaction was investigated by analyzing the structure of the JCV replication origin. The replication activities of JCV-SV40 hybrid origin plasmids were tested in cells constitutively producing either the JCV or SV40 T antigen. Results indicated that a region of the JCV origin critical for interaction with the JCV T antigen was positioned to the late side of the central palindrome of the putative core origin. A mutational analysis of this region indicated that the sequence of the A + T-rich tract was primarily responsible for determining the efficiency with which JCV can initiate replication from its origin. The tandemly repeated pentameric sequence AGGGA located proximal to the A + T-rich tract in the JCV enhancer element was found to stimulate JCV, but not SV40, T antigen-mediated replication. The effect on replication of other elements within the JCV enhancer was also dependent on the T antigen employed for initiation. A plasmid containing the replication origin of prototype BK virus was unable to replicate in cells containing JCV T antigen, again indicating the inflexibility of the JCV T antigen in interacting with heterologous origins.
Animals↗
Inactivation of DNA replication origins by the cell cycle regulator, trigonelline, in root meristems of Lactuca sativa.
The effects of trigonelline (TRG) on the cell cycle in root meristems of Lactuca sativa L. were examined in the knowledge that TRG is a cell cycle regulator that causes cell arrest in G2, and prevents ligation of replicons in S-phase. The hypothesis was tested that continuous exposure to TRG would perturb DNA replication which, in turn, would lengthen the cell cycle and impair root elongation. Using DNA fibre autoradiography, mean replicon size was 31 and 13 microm in the TRG (3 mM) and control treatments, respectively. Trigonelline also resulted in a lengthening of both S-phase and the cell cycle and a decrease in primary root elongation. Hence, replicon inactivation was responsible for the protracted S-phase. Trigonelline treatment also resulted in a 1.6-fold increase in fork rate (13.8 microm h(-1)) compared with the control (8.4 m h(-1)). The faster fork rate in the larger replicons is in accord with the highly significant positive relationship already established between fork rate and replicon size for various unrelated higher plants.
Alkaloids↗
Monomerization of RepA dimers by heat shock proteins activates binding to DNA replication origin.
DnaK is a major heat shock protein of Escherichia coli and the homolog of hsp70 in eukaryotes. We demonstrate the mechanism by which DnaK and another heat shock protein, DnaJ, render the plasmid P1 initiator RepA 100-fold more active for binding to the P1 origin of replication. Activation is the conversion of RepA dimers into monomers in an ATP-dependent reaction and the monomer form binds with high affinity to oriP1 DNA. Reversible chemical denaturants also convert RepA dimers to monomers and simultaneously activate oriP1 DNA binding. Increasing protein concentration converts monomers to dimers and deactivates RepA. Based on our data and previous work, we present a model for heat shock protein action under normal and stress conditions.
Adenosine Triphosphate↗
Cloning and characterization of oriL2, a large palindromic DNA replication origin of herpes simplex virus type 2.
An origin of replication within the long unique sequence of herpes simplex virus type 2 designated oriL2 has been identified in a position homologous to its type 1 counterpart, oriL1, between map coordinates 0.398 and 0.413. The difficulties encountered in previous attempts to clone both oriL2 and oriL1 in an undeleted form were surmounted by minimizing the growth of the host Escherichia coli, using a recBC sbcB E. coli host, and purifying the full-length plasmid from delected forms by using a novel method which exploits the ability of a palindrome-containing plasmid to adopt a cruciform conformation, thereby decreasing its supercoiling. In a previously developed assay for functional origin activity, oriL2 was localized to a 241-base-pair ApaI-SstII fragment. DNA sequence analysis revealed a 136-base pair, almost perfect palindrome. Comparison with oriL1 showed a very high degree of conservation: the two origins differ in only 16 of the 144-base-pair oriL1 palindromic region. Most significantly, the differences between oriL1 and oriL2 mainly occur in pairs so as to generally preserve the potential for intrastrand base pairing. The central region of oriL2 is homologous with the shorter palindromic structures detected in origins located within the repetitive sequences of the short component of herpes simplex virus type 1 or 2.
Chromosome Deletion↗
Two spatially distinct genetic elements constitute a bipartite DNA replication origin in the minute virus of mice genome.
Mutations were introduced into plasmid pMM984, a full-length infectious clone of the fibrotropic strain of minute virus of mice, to identify cis-acting genetic elements required for the excision and replication of the viral genome. The replicative capacity of these mutants was measured directly, using an in vivo transient DNA replication assay following transfection of plasmids into murine A9 cells and primate COS-7 cells. Experiments with subgenomic constructs indicated that both viral termini must be present on the same DNA molecule for replication to occur and that the viral nonstructural protein NS-1 must be provided in trans. The necessary sequences were located within 1,084 and 807 nucleotides of the 3' and 5' ends of the minute virus of mice genome, respectively. The inhibitory effect of deletions within the 206-bp 5'-terminal palindrome demonstrated that these sequences comprise a cis-acting genetic element that is absolutely essential for the excision and replication of viral DNA. The results further indicated a requirement for a stem-plus-arms T structure as well as for the formation of a simple hairpin. In addition, the removal of one copy of a tandemly arranged 65-bp repeat found 94 nucleotides inboard of the 5'-terminal palindrome inhibited viral DNA replication in cis by 10- and just greater than 100-fold in A9 and COS-7 cells, respectively. The latter results define a novel genetic element within the 65-bp repeated sequence, distinct from the terminal palindrome, that is capable of regulating minute virus of mice DNA replication in a species-specific manner.
Animals↗
New pUC-derived cloning vectors with different selectable markers and DNA replication origins.
Four new Escherichia coli cloning vectors are described, pUC6S, pUC21, pUK21 and pOK12. These vectors contain a polylinker or multiple cloning site (MCS) with the recognition sequences for 28 restriction enzymes. Plasmids pUC21, pUK21, and pOK12 contain the MCS in the N-terminal end of the lacZ alpha fragment allowing blue/white screening for inserts. To potentially increase the stability of some inserts that may encode toxic proteins, the strength of the lacZ promoter present on these vectors has been reduced. Plasmids pUC6S and pUC21 carry the bla gene encoding ampicillin resistance, while pUK21 and pOK12 contain the gene encoding kanamycin resistance. Plasmid pOK12 carries the replicon from P15A, resulting in a lower copy number pUC-type vector. Plasmid pUC6S carries the ori and bla gene present on all pUC vectors, but does not contain any lac sequences. Plasmids pUC21 and pUK21 contain the M13 intergenic region allowing for the production of plasmid single-stranded DNA. To improve the yield of ss plasmid DNA, two plasmid cis-acting factors that affect yield were also examined: the effect of plasmid-derived transcription across the M13 ori, and the effect of delecting the M13 minus-strand ori from the plasmid.
Base Sequence↗
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.
Adenoviruses, Human↗
EBNA1 can link the enhancer element to the initiator element of the Epstein-Barr virus plasmid origin of DNA replication.
The plasmid origin of DNA replication of Epstein-Barr virus, oriP, is replicated once per cell division, employing cellular replication machinery and only one viral protein. To understand how replication from this origin is initiated and regulated, we purified this viral protein, EBNA1. EBNA1 was expressed in CV-1p cells by using an infectious simian virus 40 vector containing the EBNA1 gene. It was purified in two chromatographic steps to apparent homogeneity. The purified protein is capable of supporting transcription of the luciferase gene from a reporter plasmid carrying the FR enhancer element to which EBNA1 binds. EBNA1 does not have oriP-dependent ATPase activity, indicating that it does not carry out an energy-dependent step in the initiation of DNA replication. However, EBNA1 does mediate an association between the two elements of oriP. We measured this association by binding one of the elements, the enhancer element, to a solid matrix and measuring retention by this element of the other one, the initiator element, in the presence of EBNA1. This retention is specific for DNA fragments containing EBNA1-binding sites. EBNA1 thus can link the two elements of the origin, providing a locally high concentration of EBNA1 at the site of initiation of DNA replication. We propose that this association is important either (i) to affect DNA structure to allow a cellular helicase to initiate DNA strand separation or (ii) to bind replication proteins to bring them to the origin of replication.
Adenosine Triphosphatases↗
High-resolution mapping of the origin of DNA replication in the hamster dihydrofolate reductase gene domain by competitive PCR.
By the use of a highly sensitive mapping procedure allowing the identification of the start sites of DNA replication in single-copy genomic regions of untreated, exponentially growing cultured cells (M. Giacca, L. Zentilin, P. Norio, S. Diviacco, D. Dimitrova, G. Contreas, G. Biamonti, G. Perini, F. Weighardt, S. Riva, and A. Falaschi, Proc. Natl. Acad. Sci. USA 91:7119-7123, 1994), the pattern of DNA replication of the Chinese hamster dihydrofolate reductase (DHFR) gene domain was investigated. The method entails the purification of short stretches of nascent DNA issuing from DNA replication origin regions and quantification, within this sample, of the abundance of different adjacent segments by competitive PCR. Distribution of marker abundance peaks around the site from which newly synthesized DNA had emanated. The results obtained by analysis of the genomic region downstream of the DHFR single-copy gene in asynchronous cultures of hamster CHO K1 cells are consistent with the presence of a single start site for DNA replication, located approximately 17 kb downstream of the gene. This site is coincident with the one detected by other studies using different techniques in CHO cell lines containing an amplified DHFR gene domain.
Animals↗
Phosphorylation-dependent interaction of adenovirus preterminal protein with the viral origin of DNA replication.
Adenovirus preterminal protein (pTP) exists as a heterodimer with the viral DNA polymerase (AdPol) and becomes covalently linked to a dCMP residue during initiation of DNA replication. The in vivo phosphorylation of pTP could be demonstrated when pTP is overproduced using recombinant vaccinia viruses, or by a large scale metabolic labeling of adenovirus 2 (Ad2)-infected HeLa cells. Phosphoserine was the only phosphoamino acid obtained by acid hydrolysis of 32P-labeled pTP immunoprecipitated from metabolically labeled HeLa cells infected with either Ad2 or recombinant vaccinia virus. Tryptic peptide maps of pTP expressed using recombinant vaccinia virus system in HeLa cells revealed that phosphorylation of pTP occurred on multiple sites. Dephosphorylation of pTP with calf intestinal alkaline phosphatase resulted in a significant decrease in its activity in the in vitro DNA replication initiation assays. Further characterization of the phosphatase-treated pTP indicated that although dephosphorylation did not affect its interaction with AdPol, the specific recognition of the DNA replication origin by pTP was significantly reduced as determined by gel electrophoresis-based DNA mobility shift assays.
Adenoviruses, Human↗
Two steps to binding replication origins? DNA-protein interactions.
Recent structural studies of the Epstein-Barr virus EBNA1 protein bound to DNA suggest that it binds to DNA replication origins in a two-step process; the first step involves recognition of the correct sequence and the second initiates structural changes in the DNA.
DNA↗