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At least 73 records · Page 4Linked to original sources

PubMed1992

The simian virus 40 T antigen double hexamer assembles around the DNA at the replication origin.

An initial step in the replication of simian virus (SV40) DNA is the ATP-dependent formation of a double hexamer of the SV40 large tumor (T) antigen at the SV40 DNA replication origin. In the absence of DNA, T antigen assembled into hexamers in the presence of magnesium and ATP. Hexameric T antigen was stable and could be isolated by glycerol gradient centrifugation. The ATPase activities of hexameric and monomeric T antigen isolated from parallel glycerol gradients were identical. However, while monomeric T antigen was active in the ATP-dependent binding, untwisting, unwinding, and replication of SV40 origin-containing DNA, hexameric T antigen was inactive in these reactions. Isolated hexamers incubated at 37 degrees C in the presence of ATP remained intact, but dissociated into monomers when incubated at 37 degrees C in the absence of ATP. This dissociation restored the activity of these preparations in the DNA replication reaction, indicating that hexameric T antigen is not permanently inactivated but merely assembled into a nonproductive structure. We propose that the two hexamers of T antigen at the SV40 origin assemble around the DNA from monomer T antigen in solution. This complex untwists the DNA at the origin, melting specific DNA sequences. The resulting single-stranded regions may be utilized by the T antigen helicase activity to initiate DNA unwinding bidirectionally from the origin.

Adenosine Triphosphate↗

PubMed1986

Characterization of a defective phage system for the analysis of bacteriophage T4 DNA replication origins.

We have developed a defective phage system for the isolation and analysis of phage T4 replication origins based on the T4-mediated transduction of plasmid pBR322. During the initial infection of a plasmid-containing cell, recombinant plasmids with T4 DNA inserts are converted into fully modified linear DNA concatamers that are packaged into T4 phage particles, to create defective phage (transducing particles). In order to select T4 replication origins from genomic libraries of T4 sequences cloned into the plasmid pBR322, we searched for recombinant plasmids that transduce with an unusually high efficiency, reasoning that this should select for T4 sequences that function as origins on plasmid DNA after phage infection. We also selected for defective phage that can propagate efficiently with the aid of a coinfecting helper phage during subsequent rounds of phage infection, which should select for T4 sequences that can function as origins on the linear DNA present in the defective phage. Several T4 inserts were isolated repeatedly in one or both of these selective procedures, and these were mapped to particular locations on the T4 genome. When plasmids were selected in this way from genomic libraries constructed using different restriction nucleases, they contained overlapping segments of the T4 genome, indicating that the same T4 sequences were selected. The inserts in two of the selected plasmids permit a very high frequency of transduction from circular plasmids; these have been shown to contain a special type of T4 replication origin.

DNA Replication↗

PubMed1985

A defective phage system reveals bacteriophage T4 replication origins that coincide with recombination hot spots.

Plasmid transduction mediated by bacteriophage T4 has been used to study putative T4 DNA replication origins cloned as inserts in the Escherichia coli plasmid pBR322. Two particular inserts from the T4 genome allow high-frequency plasmid transduction, suggesting that each insert might contain a T4 replication origin. T4 infection of these plasmid-containing cells produces large numbers of defective phage particles that contain long linear concatamers of the plasmid DNA. During a second cycle of infection, these defective phage genomes can be replicated better than normal phage chromosomes present in the same infected cell; consequently, the T4 DNA inserts must be functioning as replication origins. Both of these origins appear to utilize a previously unrecognized mode of T4 replication initiation. Moreover, each origin coincides with a major recombination hot spot in the phage genome, and therefore this mode of replication initiation seems to involve a local stimulation of homologous genetic recombination. From a purely practical standpoint, additional DNA fragments can be cloned in an origin-containing plasmid, allowing isolation of large amounts of any DNA sequence with the glucosylated hydroxymethylcytosine modifications of T4 DNA.

DNA Replication↗

PubMed1986

Binding of purified wild-type and mutant pi initiation proteins to a replication origin region of plasmid R6K.

The three replication origins of the antibiotic resistance plasmid R6K require for their activity in Escherichia coli a DNA segment containing seven 22 base-pair direct repeats and a plasmid-encoded initiation protein (pi). The pi protein functions in the negative control of R6K replication, in addition to its requirement for the initiation of replication. Construction of a plasmid containing the pi structural gene (pir) downstream from the inducible pR promoter of bacteriophage lambda provided high levels of production of pi protein in E. coli. The pi protein was purified and shown to possess general DNA binding properties with a preference for DNA fragments containing the gamma origin of replication, the operator region of the pir gene and the R6K beta-origin region. Velocity sedimentation analysis indicates that the pi protein exists as a dimer in its native form. Agarose gel electrophoresis analysis of pi-gamma-origin complexes suggests that one pi dimer binds to each copy of the 22 base-pair direct repeats in the gamma origin region. Purified mutant pi protein obtained from a temperature-sensitive initiation mutant (pir 105-ts) exhibited temperature-sensitive binding activity to the gamma-origin region, whereas two mutant proteins exhibiting a high copy number phenotype were unaltered (pir104-cop) or slightly reduced (pir1-cop) in binding activity. The patterns of DNase I protection and enhancement were similar for the wild-type and mutant proteins examined.

Binding Sites↗

PubMed1986

Xrep, a plasmid-stimulating X chromosomal sequence bearing similarities to the BK virus replication origin and viral enhancers.

The human X chromosome-linked fragment, "Xrep," was sequenced because it exerts a positive effect on plasmid growth in both E. coli and Saccharomyces cerevisiae. The sequence revealed three features similar to the human BK virus replication origin: Xrep has a true palindrome, CCTCC(T)3CCTCC, which is similar to "true" palindrome-like sequences found at the replication origins of polyoma [CCTC(T/C)10CTCC], BK [CCTC(A/G)8CCTCC] and SV40 [CCTCC(A)6GCCTCC] viruses. Twenty nucleotides away from the true palindrome, Xrep has the sequence GAATCCTATTCACTTTT while BK virus, the human analogue of SV40, has GAAATCCCTATTCTTTT in exactly the same position relative to the true palindrome. These two 17-mers differ only in the positions of two nucleotides comparing Xrep and BK virus. Also similar to the replication origins of DNA viruses, Xrep appears to have a cluster of enhancers adjacent to the origin-like sequences. Potent enhancer-like activity was detected in pSV1 X CAT/Xrep constructs. Xrep may originate from an endogenous virus, or from an X chromosomal replication origin.

Acetyltransferases↗

PubMed1988

Localization of replication origins in pea chloroplast DNA.

The locations of the two replication origins in pea chloroplast DNA (ctDNA) have been mapped by electron microscopic analysis of restriction digests of supercoiled ctDNA cross-linked with trioxalen. Both origins of replication, identified as displacement loops (D-loops), were present in the 44-kilobase-pair (kbp) SalI A fragment. The first D-loop was located at 9.0 kbp from the closest SalI restriction site. The average size of this D-loop was about 0.7 kbp. The second D-loop started 14.2 kbp in from the same restriction site and ended at about 15.5 kbp, giving it a size of about 1.3 kbp. The orientation of these two D-loops on the restriction map of pea ctDNA was determined by analyzing SmaI, PstI, and SalI-SmaI restriction digests of pea ctDNA. One D-loop has been mapped in the spacer region between the 16S and 23S rRNA genes. The second D-loop was located downstream of the 23S rRNA gene. Denaturation mapping of recombinants pCP 12-7 and pCB 1-12, which contain both D-loops, confirmed the location of the D-loops in the restriction map of pea ctDNA. Denaturation-mapping studies also showed that the two D-loops had different base compositions; the one closest to a SalI restriction site denatured readily compared with the other D-loop. The recombinants pCP 12-7 and pCB 1-12 were found to be highly active in DNA synthesis when used as templates in a partially purified replication system from pea chloroplasts. Analysis of in vitro-synthesized DNA with either of these recombinants showed that full-length template DNA was synthesized. Recombinants from other regions of the pea chloroplast genome showed no significant DNA synthesis activity in vitro.

Chloroplasts↗

PubMed1984

Gene A protein cleavage of recombinant plasmids containing the phi X174 replication origin.

Synthetic oligonucleotides, DNA ligase and DNA polymerase were used to construct double-stranded DNA fragments homologous to the first 25, 27 or 30 b.p. of the origin of replication of bacteriophage phi X174 (nucleotides 4299-4328 of the phi X174 DNA sequence). The double-stranded DNA fragments were cloned into the unique SmaI or HindIII restriction sites in the kanamycin-resistance gene of pACYC177 (AmpR, KmR). Recombinant plasmids were picked up by colony hybridization. DNA sequencing showed that not only recombinant plasmids with the expected insert were formed, but also recombinant plasmids with a shorter insert. Recombinant plasmids with an insert homologous to the first 24, 25, 26, 27, 28 or all 30 b.p. of the phi X174 origin region were thus obtained. Supercoiled plasmids containing a sequence homologous to the first 27, 28 or 30 b.p. of the phi X174 origin region are nicked by the phi X174 gene A protein. However, the other supercoiled plasmids are not nicked by the phi X174 gene A protein. These results show that the first 27 b.p. of the phi X174 origin region are sufficient as well as required for the initiation step in phi X174 RF DNA replication, i.e. the cleavage by gene A protein.

Bacteriophage phi X 174↗

PubMed1985

Conservation of genes and their organization in the chromosomal replication origin region of Bacillus subtilis and Escherichia coli.

The organization of six open reading frames which were deduced from the nucleotide sequence of some 10 kb from the replication origin region of Bacillus subtilis resembles the organization of the genes in the rnpA-dnaA-gyrB region of the Escherichia coli chromosome. Based on the detection of homology with the E. coli genes the open reading frames were found to represent the Bacillus 'rnpA', 'rpmH', 'dnaA', 'dnaN', recF and gyrB genes. Only the latter two have also been defined by genetic analysis. Two regulatory regions containing nine and four copies of a repeating sequence, DnaA-box, which is identical with the DnaA protein-binding sequence repeated four times in the E. coli origin of replication, flank the 'dnaA' gene of B. subtilis. One or both of them are proposed to function as origins in the initiation of chromosomal replication. Transcription of the 'dnaA' gene of Bacillus starts in one of these regions and appears to be coupled to initiation of chromosomal replication. We propose that the conserved gene organization in the 'dnaA'-'gyrB' region of B. subtilis is representative of the replication origin region of a primordial replicon. The oriC sequence of E. coli has either been translocated to its present location 44 kb away from the primordial origin or has independently evolved there.

Amino Acid Sequence↗

PubMed1984

oriX: a new replication origin in E. coli.

Replication of the chromosome of E. coli at 42 degrees C in an integratively suppressed dnaA mutant (dnaA46 Sin Hfr) occurs predominantly from the origin of replication of the integrated plasmid (oriV). We have carried out a detailed marker frequency analysis on such Hfrs. This analysis indicates that replication at 42 degrees C occurs not only from oriV, but also from an origin, oriX, located in the terminal region of the chromosome close to, but distinct from, the prophage rac (oriJ). In an oxa1 mutant of one of these Hfrs, we have shown that replication proceeds at 42 degrees C from all three origins: oriV, oriX, and oriC. Loss of the integrated plasmid results in a temperature- and rich-medium-sensitive strain that replicates the chromosome from oriC and oriX. Replication from oriX proceeds slowly and bidirectionally. We suggest that oriX may be involved in the coupling between replication and cell division.

Bacterial Proteins↗

PubMed1979

The nucleotide sequence of a DNA fragment from the replication origin of the antibiotic resistance factor R1drd19.

The recombinant plasmid pRK101 contains a DNA fragment which carries the complete replication origin of the antibiotic resistance factor R1drd-19 inserted into the vector plasmid pBR322. In a spontaneously arising mutant of this plasmid (pRK103) a deletion of about 215 base pairs (bp) has been detected by heteroduplex analysis and mapping with restriction endonucleases. Essential parts of the replication origin must be located in the deleted sequence. The deletion mutant pRK103, in contrast to its parent plasmid pRK101 is not replicated under the control of the R1 replicon, even when the R1 factor or copy mutants of it are present within the same cell. These latter plasmids can complement a plasmid-specific protein not coded by pRK101 but essential for R1-directed replication. The nucleotide sequence of a 252 bp HpaII fragment covering about 170--200 bp of the deletion was determined. This piece of DNA is rich in G and C and contains a series of small palindromes, symmetrically arranged repeated sequences and short selfcomplementary structures which may be of significance for the initiation of the DNA replication. The possiblity that the sequenced DNA fragment comprises a major part of the replication origin of R1drd-19 is discussed.

Base Sequence↗

PubMed1984

Direct evidence for specific binding of the replicative origin of the Escherichia coli chromosome to the membrane.

The origin of replication of the Escherichia coli chromosomal DNA binds with high affinity to outer membrane preparations. This specific binding requires a 463-base-pair region of origin DNA between positions -45 and +417 of the oriC map. We show that binding does not require the presence of adjacent regions. From further analysis, we conclude that more than one binding site resides within the 325-base-pair fragment between positions +38 (BamHI) and +417 (XhoI). When this fragment is cut, two pieces bind with high affinity and one binds with lesser affinity. The binding ability of one of the high affinity sites is abolished by cutting it at position +92 with BamHI.

Binding Sites↗

PubMed1989

Localization of the replication origin of plasmid pE194.

The pE194 replication origin was localized to a 265-base-pair interval by analyzing the ability of purified pE194 restriction fragments to direct replication of heterologous plasmids. Replication was dependent upon RepF protein supplied in trans. The origin region contained a GC-rich dyad symmetry which may serve as the RepF target.

Bacillus subtilis↗

PubMed1991

Cooperative assembly of simian virus 40 T-antigen hexamers on functional halves of the replication origin.

The cofactor ATP stimulates the formation of T-antigen double hexamers on the simian virus 40 core origin of replication (I. A. Mastrangelo, P. V. C. Hough, J. S. Wall, M. Dodson, F. B. Dean, and J. Horwitz, Nature [London] 338:658-662, 1989). We report here the pathway for the assembly of hexamers and double hexamers on the core origin. ATP triggers the cooperative assembly of hexamers on the early and late halves of the origin even when they are completely isolated. Hexamer assembly nucleates at T-antigen recognition pentanucleotides in the early half of the origin. In intact origins, assembly of the first hexamer on the early half of the origin cooperatively stimulates the assembly of a second hexamer on the adjacent late half of the origin. Thus, monomer-monomer and hexamer-hexamer interactions of T antigen, allosterically activated by ATP, constitute two distinct types of cooperative interaction with the origin. Finally, we show that the assembly of T-antigen hexamers on isolated half origins leads to the same array of structural changes that T antigen induces in intact origins. We conclude that the origin is divided into complementary halves that each promote the assembly of functional T-antigen hexamers.

Adenosine Triphosphate↗

PubMed1986

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↗

PubMed1986

Replication origins are attached to the nuclear skeleton.

DNA fragments containing replication origins (oriDNA) were isolated from a chicken erythroblast cell line by a modified procedure of Zannis-Hadjopoulos et al. and studied in the renaturation reaction driven by either total or nuclear matrix DNA (nmDNA) from the same cells or from mature erythrocytes. We found that the unique sequences of nmDNA from erythroblasts (5 kb long) represented a specific subset of sequences constituting about a quarter of total DNA unique sequences, while the erythrocyte nmDNA 5 kb fragments constitute only about one tenth of total unique DNA and all are recovered among erythroblast nmDNA. Virtually all oriDNA sequences are present in the fraction of erythrocyte nmDNA. Thereafter, the putative positions of replication origins within the alpha-globine gene domain have been mapped by hybridization experiments. They were found to coincide with the previously established positions of permanent sites of DNA attachment to the nuclear matrix.

Animals↗

PubMed1985

DNA-protein interaction at the replication origins of plasmid chromosomes.

Novel techniques have been developed to purify replication initiator proteins of the plasmids R6K and pSC101. The techniques consist of tagging the initiator cistrons at the C-terminus with beta-galactosidase-encoding DNA of Escherichia coli in the correct translational phase. The hybrid proteins are then rapidly purified by adsorption to and elution from a beta-galactosidase- specific affinity column. Two procedures have been devised to isolate the nonfused initiator proteins using the fused protein as a handle. The first procedure, called subunit association chromatography, exploits the association of a monomer of nontagged protein with that of beta-galactosidase-tagged protein in isolating both types of proteins by beta-galactosidase specific affinity column chromatography. The second procedure involves the fusion of the initiator protein to beta-galactosidase via a specific linker DNA. The linker DNA encodes a protein which is readily and specifically hydrolyzed by a sequence specific protease, thus releasing the initiator protein from beta-galactosidase. Using purified or partially purified initiator protein, we have demonstrated that the R6K encoded initiator protein (Pi protein) binds to a consensus 22 bp sequence at 2 regions of the plasmid chromosome. The pSC101-encoded initiator protein binds to sequences at or near the plasmid replication origin. At low concentrations the protein binds to a nucleation site and upon raising the concentrations of the protein binding is promoted at 4 adjacent sequences that have partial homologies with the nucleation sequence. Deletion of the binding site leads to a nonfunctional replication origin.

DNA Replication↗

PubMed1991

The sequence-directed bent DNA detected in the replication origin of Chlamydomonas reinhardtii chloroplast DNA is important for the replication function.

We demonstrated that the 1055 bp restriction fragment containing OriA, a chloroplast DNA replication origin of Chlamydomonas reinhardtii, has electrophoretic anomalies characteristic of bent DNA. A tandem dimer of the region was constructed. Quantitative measurement of the relative gel mobility of a set of permuted fragments was used to extrapolate the approximate position of the bent DNA segment. By analyzing the gel mobility of short, sequenced fragments of the bent DNA region, the putative bending locus was identified. Two A4 tracts and two A5 tracts were located in the bending locus. Oligonucleotide-directed mutagenesis was then used to disrupt the A tract or the spacing between A tracts and the effect of site-specific mutation on electrophoretic mobility was analyzed. To assess the functional role of the bent DNA region, subclones containing the bending locus, mutated bending locus, and regions flanking the bending locus were constructed. Each subclone was used as template in an in vitro DNA replication system which preferentially initiated DNA replication at OriA. A 224 bp subclone with the bending locus positioned in the middle displayed the highest replication function and was sufficient to initiate DNA replication in vitro. Site-specific mutations or alterations of the A tracts resulted in decreased DNA bending and decreased DNA replication activity.

Base Sequence↗

PubMed1978

Map position of the replication origin on the E. coli chromosome.

Strains carrying a dnaA temperature sensitive (t.s.) mutation and a Mu-1 prophage inserted within different genes near the origin of replication have been constructed. For each strain, integratively suppressed Hfrs, named G and D in which the ori region was replicated clockwise and counterclockwise respectively, were isolated. The strand preferences of Mu-1 specific Okazaki fragments were subsequently determined for each t.s. strain and its Hfr derivatives. Their comparison led us to establish the direction of replication of the Mu-1 marker from ori. The site ori was confined to the bglB-C--rbsK-P interval.

Chromosome Mapping↗