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At least 73 records · Page 4Linked to original sources
PubMed1996

Mapping of a replication origin within the promoter region of two unlinked, abundantly transcribed actin genes of Physarum polycephalum.

We analyzed the replication of two unlinked actin genes, ardB and ardC , which are abundantly transcribed in the naturally synchronous plasmodium of the slime mold Physarum polycephalum. Detection and size measurements of single-stranded nascent replication intermediates (RIs) demonstrate that these two genes are concomitantly replicated at the onset of the 3-h S phase and tightly linked to replication origins. Appearance of RIs on neutral-neutral two-dimensional gels at specific time points in early S phase and analysis of their structure confirmed these results and further established that, in both cases, an efficient, site-specific, bidirectional origin of replication is localized within the promoter region of the gene. We also determined similar elongation rates for the divergent replication forks of the ardC gene replicon. Finally, taking advantage of a restriction fragment length polymorphism, we studied allelic replicons and demonstrate similar localizations and a simultaneous firing of allelic replication origins. Computer search revealed a low level of homology between the promoters of ardB and ardC and, most notably, the absence of DNA sequences similar to the yeast autonomously replicating sequence consensus sequence in these Physarum origin regions. Our results with the ardB and ardC actin genes support the model of early replicating origins located within the promoter regions of abundantly transcribed genes in P. polycephalum.

Actins↗
PubMed1996

Composite patterns in neutral/neutral two-dimensional gels demonstrate inefficient replication origin usage.

The neutral/neutral two-dimensional (2-D) gel replicon mapping technique has been used to great advantage to localize and characterize origins of replication. Interestingly, many yeast origins display a composite pattern consisting of both a bubble arc and a single-fork arc. Moreover, in every instance in which neutral/neutral 2-D gels have been used to analyze origins in higher eukaryotic cells, two or more adjacent fragments display these composite patterns. We believe that composite patterns signal inefficient origin usage in yeast cells because the replicators in question are not active in every cell cycle and in higher eukaryotic replicons because initiation sites are chosen from among many potential sites lying within a zone. However, others have suggested that the single-fork arcs in these composite gel patterns arise from nicking activity that converts replication bubbles to branched structures that comigrate with bona fide single forks. Here, we have used three different replicon mapping strategies to show that broken simian virus 40 replication bubbles trace unique arcs that are clearly distinguishable from classic, intact single forks. Thus, it is likely that composite 2-D gel patterns represent origins that are inefficiently utilized.

Animals↗
PubMed1977

Replication process of the parvovirus H-1. VIII. Partial denaturation mapping and localization of the replication origin of H-1 replicative-form DNA with electron microscopy.

Partial denaturation mapping, restriction endonuclease digestion, and electron microscopy were used to determine which end of the linear duplex replicative-form (RF) DNA molecule contains the origin of RF replication for the parvovirus H-1. This origin was localized within approximately 300 base pairs of the arbitrarily designated right end of the RF DNA, in the EcoRI or HaeII-A fragment. Based on denaturation behavior in formamide, the right end was also found to have a relatively high guanine plus cytosine content, whereas the region adjacent to the left terminus of the RF DNA molecule was adenine plus thymine rich.

DNA Replication↗
PubMed1974

More precise mapping of the replication origin in Escherichia coli K-12.

The origin of replication in Escherichia coli K-12 was mapped by determining the rate of marker replication during a synchronous round of replication. Four isogenic strains were made lysogenic for lambdaind(-) and for phage Mu-1, with Mu-1 integrated into a different chromosomal location in each strain. Cultures were starved for amino acids to allow completion of chromosome replication cycles and then starved for thymine in the presence of amino acids, and a synchronous cycle of replication was initiated by the addition of thymine. Samples were exposed to radioactive thymidine at intervals, deoxyribonucleic acid was extracted, and the rate of marker replication was determined by deoxyribonucleic acid-deoxyribonucleic acid hybridization to filters containing Mu-1, lambda, and E. coli deoxyribonucleic acid. The results confirm that the origin of replication is near ilv. The travel times of the replication forks, calculated from the data obtained for cultures with doubling times of approximately 40 and 61 min, are 40 and 52 min, respectively.

Amino Acids↗
PubMed1972

Deoxyribonucleic acid-deoxyribonucleic acid hybridization assay for replication origin deoxyribonucleic acid of Escherichia coli.

Deoxyribonucleic acid (DNA)-DNA hybridization on nitrocellulose filters can be used to assay for replication origin DNA from Escherichia coli if the DNA attached to the filters is enriched for the replication origin sequences. Such DNA can be readily isolated from very rapidly growing cells. When low amounts of this DNA were attached to filters, radioactively labeled DNA from the replication origin hybridized 1.7 times as well as radioactive replication terminus DNA. Under identical conditions, radioactively labeled DNA from exponentially growing cells hybridized only 1.3 times as well as radioactive replication terminus DNA. The replication origin, replication terminus, and randomly labeled DNA hybridized with similar efficiencies to filters containing DNA isolated from cells incubated in the absence of required amino acids. This DNA appeared to have all sequences present at equal frequencies. The hybridization assay was used to demonstrate that the DNA synthesized shortly after the addition of amino acids to cells previously deprived of required amino acids was primarily from the replication origin and then rapidly became similar to DNA synthesized by exponentially growing cells.

Amino Acid Sequence↗
PubMed1997

Interaction of P1 RepA with replication origin of plasmid Rts1: capability of an initiator protein inducing replication from a foreign origin.

Rts1 RepA and P1 RepA are trans-acting proteins essential for the initiation of replication of plasmid Rts1 and prophage P1, respectively. In this study, we found that, in vitro, P1 RepA bound to the Rts1 ori fragment and Rts1 incI fragment as strongly as Rts1 RepA. In addition P1 RepA, in trans, activated the Rts1 replication origin that was cloned in pBR322, thus allowing the ori plasmid to be maintained in a polA E. coli host. Under these conditions, however, the ori plasmid was unstable as compared with that when activated by Rts1 RepA. In addition, we found that Rts1 RepA showed no interaction with the P1 replication origin.

Bacteriophage P1↗
PubMed1998

The replication inhibition activity of the WT1 tumor suppressor protein resides in its N-terminal 298 amino acid region, and does not require specific binding of the protein to the replication origin sequence.

Using the simian virus 40 replication origin as the model, it was reported previously that the Wilms' tumor suppressor protein WT1 can inhibit DNA replication. In the present study, we found that a hybrid protein (termed GAL4-WT1AE Z) consisting of the DNA-binding domain of the yeast transcription factor GAL4 fused in-frame with the N-terminal 298 amino acid (aa) transcriptional regulatory region of WT1 retained the ability to inhibit replication. The hybrid protein and the full-length WT1 inhibited replication without regard to the presence or absence of their binding sites in the replication origin region, indicating that inhibition of replication by the proteins does not require their specific binding to the origin region. The inhibition efficiency of the hybrid protein was the same as that of WT1, indicating that the replication inhibition activity resides in the N-terminal 298 aa region, and that the C-terminal zinc finger-containing DNA-binding domain of WT1 is functionally dispensable for this effect.

Amino Acid Sequence↗
PubMed1996

Common structural features of replication origins in all life forms.

Origins of replication (ORIs) among prokaryotes, viruses, and multicellular organisms appear to possess simple tri-, tetra-, or higher dispersed repetitions of nucleotides, AT tracts, inverted repeats, one to four binding sites of an initiator protein, intrinsically curved DNA, DNase I-hypersensitive sites, a distinct pattern of DNA methylation, and binding sites for transcription factors. Eukaryotic ORIs are sequestered on the nuclear matrix; this attachment is supposed to facilitate execution of their activation/deactivation programs during development. Furthermore, ORIs fall into various classes with respect to their sequence complexity: those enriched in AT tracts, those with GA- and CT-rich tracts, a smaller class of GC-rich ORIs, and a major class composed of mixed motifs yet containing distinct AT and polypurine or GC stretches. Multimers of an initiator protein in prokaryotes and viruses that might have evolved into a multiprotein replication initiation complex in multicellular organisms bind to the core ORI, causing a structural distortion to the DNA which is transferred to the AT tract flanking the initiator protein site; single-stranded DNA-binding proteins then interact with the melted AT tract as well as with the DNA polymerase alpha-primase complex in animal viruses and mammalian cells, causing initiation in DNA replication. ORIs in mammalian cells seem to colocalize with matrix-attached regions and are proposed to become DNase I-hypersensitive during their activation.

Animals↗
PubMed1986

A replication origin is turned off by an origin-"silencer" sequence.

The chromosome of R6K contains multiple origins of replication. The origin gamma is infrequently used in the original plasmid and remains "silent" in certain miniplasmid derivatives. The inactivation of the origin is caused by a natural origin silencer located adjacent to the minimal ori gamma sequence. The silencer functions in cis and has no trans activity. It has functional polarity and works only in one orientation when present immediately downstream from ori gamma. The silencer apparently initiates an RNA that invades ori gamma and turns it off either by competing with a primer RNA or by disrupting ori gamma structure. As predicted, removal of the silencer blocks the synthesis of silencer RNA and derepresses the origin.

Base Sequence↗
PubMed1995

Clusters of modular regulatory elements at DNA replication origins.

To study the specificity of eukaryotic origins of replication (ori), we have isolated a fraction of mouse DNA enriched in replication initiation sequences (RIS), and cloned and characterised some of these RIS. The sequences of three RIS were analysed for the presence of sequence elements common to other known eukaryotic ori. It was found that the three RIS were A+T rich and contained a number of sequence elements that may function in the initiation of DNA replication. The data support the idea that mammalian ori are organised from modular sequence elements.

Animals↗
PubMed1994

Loading of a DNA helicase on the DNA unwinding element in the yeast replication origin: mechanism of DNA replication in a model system.

We found that initiation of DNA replication occurs from the region containing the yeast autonomously replicating sequence 1 (ARS1), by incubating negatively supercoiled plasmid DNA with the proteins required for SV40 DNA replication in addition to DNA gyrase (Ishimi, Y., & Matsumoto, K. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 5399-5403). Here, the mechanism of DNA replication and the roles of the replication proteins in this model system were analyzed. Both SV40 T antigen as a DNA helicase and multisubunit human single-stranded DNA binding protein (HSSB) (also called RP-A) were required for the initial step of DNA synthesis. Furthermore, it has been shown that T antigen plays an essential role in the initiation of DNA replication from the ARS region in this system. The digestion of negatively supercoiled DNA with the single-strand-specific nuclease P1 revealed that regions containing A, B, and C domains of ARS1 can be unwound under the conditions used for DNA replication. Footprinting with KMnO4 indicated that T antigen interacted with the unwound B domain where initiation of DNA replication mainly occurred. When circular DNAs of different negative-superhelical densities were replicated in the absence of DNA gyrase, short fragments were synthesized from the ARS region in proportion to its density and they were elongated by addition of HeLa topoisomerase I, which inhibits the initiation of DNA replication in this system.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigens, Polyomavirus Transforming↗
PubMed1984

Chromatin structure at the replication origins and transcription-initiation regions of the ribosomal RNA genes of Tetrahymena.

The chromatin structure of regulatory regions of the extrachromosomal rRNA genes of Tetrahymena thermophila was probed by nuclease treatment of isolated nuclei. The chromatin near the origins of replication contains hypersensitive sites for micrococcal nuclease, DNAase I, and DNAase II. These sites persist in starved cells, consistent with the origins' being maintained in an altered chromatin structure independent of DNA replication. The region between the two origins of replication is organized into a phased array of seven nucleosomes, the fourth of which is centered at the axis of symmetry of the palindromic rDNA. The entire transcribed region and 150 bp upstream from the initiation site are generally accessible to nucleases; any histone proteins associated with these regions are clearly not in a highly organized nucleosomal array as seen in the central region. Comparison of the chromatin structures of the central spacer of T. thermophila and T. pyriformis rDNA reveals that deletion or insertion of DNA has occurred in increments of 200 bp. This is taken to imply that there are constraints on the evolution of spacer DNA sequences at the level of the nucleosome.

Animals↗
PubMed1986

Permanent attachment of replication origins to the nuclear matrix in BHK-cells.

The position of replication origins and replication forks relative to the nuclear matrix was analysed by autoradiography. Analysis of 2M NaCl-extracted extracted BHK-nuclei, prepared on coverslips, showed that after brief pulses grains were exclusively found over the central core of the residual nuclei, which corresponds to positions in the nuclear matrix. In asynchronous cells these grains were found to migrate into the DNA-halo surrounding the matrix during a subsequent chase. When the pulse had been administered to synchronous cells at the onset of S-phase, it was observed, however, that in the majority of the structures no such migration had occurred. From this, and from the fact that label incorporated later in S-phase could be chased into the halo, we conclude that, contrary to DNA in replication forks, DNA containing replication origins is permanently attached to the nuclear matrix.

Animals↗
PubMed1998

Characterization of a plasmid replicative origin from an extreme thermophile.

The minimal replicative origin from a 16-kbp natural plasmid of Thermus sp. ATCC 27737 is described. The 1798-bp sequence encodes a single protein (RepA) whose expression is required for replication. This requirement is extended to 200 bp upstream the coding region, which contains promoter-like sequences transcriptionally active in E. coli. Despite the absence of typical iterons, we show that an N-terminal fragment of this protein binds to a DNA fragment internal to its own coding sequence. As this DNA fragment contains an unusually A + T rich sequence surrounded by C + G rich stretches, the possible location of the replicative origin within the region encoding RepA is proposed.

Amino Acid Sequence↗
PubMed1994

Eukaryotic replication origins: control in space and time.

Replication origins facilitate the choreography of genome duplication by acting as targets for regulatory mechanisms. Eukaryotic cells control the efficiency and the time of origin activity. In addition, origins that have been replicated are prevented from doing so twice in the same cell cycle. In this review we will examine the mechanisms that may be used to control these processes, and discuss the role of replication in regulating transcription.

Animals↗
PubMed1980

The nucleotide sequence surrounding the replication origin of the cop3 mutant of the bacteriocinogenic plasmid Clo DF13.

The nucleotide sequence from about 100 base-pairs downstream to about 600 base pairs upstream the CloDF13 replication origin has been determined. A comparison of this sequence with the corresponding ColE1 origin sequence reveals that: The sequence at the origin of replication is conserved. There are large differences in the nucleotide sequence downstream the replication origin, whereas there is a large homology in the region of about 410 base-pairs upstream the replication origin. This conserved region might code for a largely homologous basic, arginine rich polypeptide of about 45 amino-acids, for both ColE1 and CloDF13. Although there are large differences in the primary structure of the region coding for the 100 nucleotide RNA, the secondary structure of this region seems to be conserved.

Base Composition↗
PubMed1990

Sequential initiation of lagging and leading strand synthesis by two different polymerase complexes at the SV40 DNA replication origin.

Enzymatic synthesis of DNA from the simian virus 40 origin of DNA replication has been reconstituted in vitro with eight purified components. DNA polymerase alpha-primase complex first initiates DNA synthesis at the replication origin and continues as the lagging strand polymerase. Subsequently, the DNA polymerase delta complex initiates replication on the leading strand template. Some prokaryotic DNA polymerase complexes can replace the eukaryotic polymerase delta complex. A model for polymerase switching during initiation of DNA replication is presented.

Animals↗
PubMed1988

Deletion analysis of bacteriophage T4 tertiary origins. A promoter sequence is required for a rifampicin-resistant replication origin.

The DNA sequence requirements of two T4 tertiary replication origins have been characterized by a deletion analysis of origin-containing plasmids. Maximal replication of each origin-containing plasmid required both an intact gpmotA-dependent middle-mode promoter sequence and approximately 50 base pairs of the downstream region. In contrast, gpmotA-dependent transcription from the origin promoter was found to be independent of the downstream region. The requirement for a promoter element within the tertiary origins is striking, particularly since the replication of tertiary origin-containing plasmids is resistant to the RNA polymerase inhibitor rifampicin.

Base Sequence↗