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At least 793 records · Page 44Linked to original sources
PubMed1995

Determination of sequence specificity between a plasmid replication initiator protein and the origin of replication.

Staphylococcal plasmids of the pT181 family replicate by a rolling circle mechanism, requiring the activities of a plasmid-specified Rep protein. The initiation event involves site-specific phosphodiester bond cleavage by Rep within the replication origin, ori. In vitro the Rep proteins also display type-I topoisomerase activity specific for this plasmid family. Although the single site of bond cleavage, ICR II, is conserved among all members of the pT181 family, the plasmid-specific Rep proteins are able to discriminate between family members in vivo, initiating replication only from the cognate origin. The basis of such specificity is believed to be due to a non-covalent binding interaction between Rep and a DNA sequence adjacent to the site of phosphodiester bond cleavage. Using the RepD protein specified by plasmid pC221, we present data for the physical parameters of RepD:oriD complex formation. Quantification of the relative strengths of the non-covalent interactions for different but related ori target sequences, measured by gel mobility shift experiments, has yielded data that are in accord with the known specificity of the protein in vivo. Oligonucleotide competition experiments demonstrate that this interaction is indeed attributable to the specificity determinant, ICR III. Protein-DNA crosslinking methods show that a carboxyl-terminal proteolytic fragment of RepD makes a specific interaction with the ICR III region of its cognate replication origin. Analysis of topoisomerase rates indicates that the interaction between ICR III and the carboxyl terminus of the protein is required before a productive interaction, namely the phosphodiester bond cleavage at the ICR II, can occur.

Bacterial Proteins↗
PubMed2000

Footprinting studies of specific complexes formed by RepA, a replication initiator of plasmid pCU1, and its binding site.

The basic replicon of plasmid pCU1 contains three different replication origins. Replication initiated from the oriB origin requires pCU1-encoded protein RepA. Previously, information analysis of 19 natural RepA binding sequences predicted a 20-bp sequence as a RepA binding site. Guanines contacting RepA in the major groove of DNA have also been determined. In this study, we used the missing-nucleoside method to determine all of the bases relevant to RepA binding. The importance of some thymine bases was also confirmed by a missing-thymine site interference assay. Participation of the 5-methyl groups of two thymines (at positions -6 and 7) in RepA binding was pointed out by a missing-thymine methyl site interference assay. Phosphate groups of the DNA backbone which strongly interfered with RepA binding upon ethylation were also identified. The pattern of contacting positions mapped by hydroxyl radical protection footprinting indicates that RepA binds to one face of B-form DNA. The length of the binding site was found to be 20 bp by dissociation rate measurement of complexes formed between RepA and a variety of binding sequences. The symmetry of the binding site and that of the contacting bases, particularly the reacting 5-methyl groups of two thymines, suggest that pCU1-encoded RepA may contact its site as a homodimer.

Bacterial Proteins↗
PubMed1978

Definition of the boundaries of the origin of DNA replication in simian virus 40.

We have determined by use of DNA sequencing techniques the exact location of the deletion in d1 892, a viable deletion mutant of Simian virus 40 (SV40) reported to map very near the unique replication origin or SV40. With the help of this localization we have narrowed down the boundaries of the replication origin to 85 nucleotides within the sequence of SV40.

Base Sequence↗
PubMed1983

DNA-protein interaction at the origin of DNA replication of the plasmid pSC101.

The initiation of DNA replication of the low copy number plasmid pSC101 is dependent on the dnaA initiator protein encoded by Escherichia coli. We have previously reported that the minimum essential replicon of the plasmid encodes a approximately 37.5 kd protein and that the protein is necessary along with host-encoded proteins for the replication of the plasmid chromosome. In this communication we show that the plasmid-encoded protein has sequence-specific DNA-binding activity. The protein binds cooperatively to the replication origin of pSC101. Using chemical and enzymatic probes we have determined the contact points of the protein with the DNA and the precise domain of the replication origin recognized by the 37.5 kd protein. The specificity of the DNA-protein interaction would suggest that the 37.5 kd protein may possibly function by guiding the replisome to the correct DNA sequence on the chromosome of pSC101.

Bacterial Proteins↗
PubMed2001

Antitumor drug adozelesin differentially affects active and silent origins of DNA replication in yeast checkpoint kinase mutants.

The antitumor drug adozelesin is a potent cytotoxic DNA-damaging agent. Here we determined how adozelesin affects chromosomal DNA replication at a molecular level in a yeast model system and examined the influence of checkpoint kinase genes, the human homologues of which are mutated in cancer. Analysis of replication intermediates using two-dimensional gel electrophoresis showed that adozelesin inhibited the activity of a replication origin and stalled replication fork progression through chromosomal DNA at the origin. RAD53 and MEC1 protein kinase genes, homologues of human CHK2 and ATM, respectively, regulate an intra-S-phase DNA damage checkpoint and, when mutated, permit unchecked replication of damaged DNA in S-phase. Mutations in these genes did not abrogate adozelesin-induced inhibition of origin activity and fork progression at the replication origin. However, novel replication intermediates indicative of DNA breaks were detected only in the rad53 mutant, suggesting a role for the wild-type gene in maintaining chromosome integrity in the presence of the drug. In contrast to the inhibition of the active replication origin by adozelesin, normally silent origins present in the same chromosome were activated by adozelesin in rad53 and mec1 mutant cells. Thus, an antitumor drug that damages DNA can induce an abnormal replication pattern in a chromosome by activating silent origins, depending upon defects in yeast checkpoint kinase genes, the homologues of which are mutated in cancer. Implications of an abnormal replication pattern for the epigenetic regulation of gene expression are discussed.

Antineoplastic Agents, Alkylating↗
PubMed1969

Relation of the segregative origin of chromosome replication to the origin of replication after amino acid starvation.

Cultures of Escherichia coli 15T(-) and K-12 were labeled with (3)H-thymine before, during, and after amino acid starvation. The number of labeled segregating units was measured by autoradiography of microcolonies derived from the labeled cells. In both strains, labels inserted before starvation and during starvation appeared to segregate as if incorporated into the same polynucleotide strands. However, labels inserted during and after starvation segregated as if incorporated into different polynucleotide strands. In view of previous data, it was concluded that replication after amino acid starvation originates from the region of the chromosome which serves as the origin for replication during normal growth and division.

Autoradiography↗
PubMed2004

ATP hydrolysis by ORC catalyzes reiterative Mcm2-7 assembly at a defined origin of replication.

The origin recognition complex (ORC) is a six-subunit, ATP-regulated, DNA binding protein that is required for the formation of the prereplicative complex (pre-RC), an essential replication intermediate formed at each origin of DNA replication. In this study, we investigate the mechanism of ORC function during pre-RC formation and how ATP influences this event. We demonstrate that ATP hydrolysis by ORC requires the coordinate function of the Orc1 and Orc4 subunits. Mutations that eliminate ORC ATP hydrolysis do not support cell viability and show defects in pre-RC formation. Pre-RC formation involves reiterative loading of the putative replicative helicase, Mcm2-7, at the origin. Importantly, preventing ORC ATP hydrolysis inhibits this repeated Mcm2-7 loading. Our findings indicate that ORC is part of a helicase-loading molecular machine that repeatedly assembles Mcm2-7 complexes onto origin DNA and suggest that the assembly of multiple Mcm2-7 complexes plays a critical role in origin function.

Adenosine Triphosphate↗
PubMed1993

Identification and characterization of a putative origin of DNA replication in the genome of a baculovirus pathogenic for Orgyia pseudotsugata.

A four-kilobase (kb) region (HindIII-N, map units 7.0-11.3) of the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (OpMNPV) genome was found to contain sequences that conferred upon plasmids the ability to undergo infection-dependent replication. The plasmid DNA appeared to be replicated to form high molecular weight multimers. Plasmids with deletions of up to 1.8 kb from either end of the HindIII-N region were replication competent. However, a discrete sequence, contained within the region bracketed by the deletions, capable of specifying replication was not identified. No evidence for sequence homology was found between the OpMNPV HindIII-N region and regions elsewhere in the OpMNPV genome or to putative Autographa californica MNPV (AcMNPV) replication origins. Origin-dependent plasmid replication was shown to require the presence of the OpMNPV DNA polymerase gene. The OpMNPV origin replicated poorly in AcMNPV-infected Spodoptera frugiperda cells and, conversely, a putative AcMNPV origin (hr2) replicated at low levels in OpMNPV-infected Lymantria dispar cells.

Amino Acid Sequence↗
PubMed2002

Recombination of poliovirus RNA proceeds in mixed replication complexes originating from distinct replication start sites.

Genetic recombination occurs frequently during replication of picornaviruses. To explore the intracellular site and structures involved in recombination, HeLa cells were infected with poliovirus type 1 Mahoney and type 2 Sabin. The two genomes were located by fluorescent in situ hybridization and confocal microscopy. For hybridization, type-specific fluorescent riboprobes were used to visualize the same genomic region where, in parallel, recombination was demonstrated with type-specific reverse transcription-PCR and sequencing. The hybridization analysis indicated that >85% of the replication complexes contained both type 1 and type 2 RNA sequences aligned at a lateral distance of 50 nm or less. Sequential infection of cells ruled out the possibility that the high percentage of mixed replication complexes was due to aggregation of input virus. Visualization of input genomic RNA over time showed that the viral genomes migrated to relatively few distinct, and thus presumably specific, perinuclear sites where replication started. The first recombinant RNA strands could be detected concomitantly with the onset of RNA replication. The limited number of start sites for replication may be the reason for the observed preferential formation of mixed replication complexes, each accommodating several parental RNA strands and thus allowing recombination.

Base Sequence↗
PubMed1991

Simian virus 40 large T antigen untwists DNA at the origin of DNA replication.

Simian virus 40 large tumor antigen (SV40 T antigen) untwists DNA at the SV40 replication origin. In the presence of ATP, T antigen shifted the average linking number of an SV40 origin-containing plasmid topoisomer distribution. The loss of up to two helical turns was detected. The reaction required the presence of the 64-base pair core origin of replication containing T antigen DNA binding site II; binding site I had no effect on the untwisting reaction. The presence of human single-stranded DNA binding protein (SSB) slightly reduced the degree of untwisting in the presence of ATP. ATP hydrolysis was not required since untwisting occurred in the presence of nonhydrolyzable analogs of ATP. However, in the presence of a nonhydrolyzable analog of ATP, the requirement for the SV40 origin sequence was lost. The origin requirement for DNA untwisting was also lost in the absence of dithiothreitol. The origin-specific untwisting activity of T antigen is distinct from its DNA helicase activity, since helicase activity does not require the SV40 origin but does require ATP hydrolysis. The lack of a requirement for SSB or ATP hydrolysis and the reduction in the pitch of the DNA helix by just a few turns at the replication origin distinguishes this reaction from the T antigen-mediated DNA unwinding reaction, which results in the formation of a highly underwound DNA molecule. Untwisting occurred without a lag after the start of the reaction, whereas unwound DNA was first detected after a lag of 10 min. It is proposed that the formation of a multimeric T antigen complex containing untwisted DNA at the SV40 origin is a prerequisite for the initiation of DNA unwinding and replication.

Adenosine Triphosphate↗
PubMed1993

Mammalian genomic sequences can substitute for the SV40 AT stretch in sustaining replication of the SV40 origin of replication.

The core of the SV40 origin of replication (ori) contains a stretch of adenine (A) and thymine (T) residues. This region is very conserved among the papova viruses, and is known to be extremely sensitive to mutations. So far, mutations have been found to drastically reduce, and in most cases abolish, replication. The AT stretch has been shown to be the target for several host cellular proteins that belong to the replication machinery. We reasoned that, in this light, there might exist cellular DNA sequences that can substitute for the SV40 AT stretch. To study this possibility, we digested mammalian genomic DNA and inserted the fragments instead of the SV40 AT stretch in a plasmid carrying the SV40 ori core. The resulting pool was analyzed by a 'replication trap' in CosI cells. We present evidence that there are indeed several mammalian sequences that can substitute for the SV40 AT stretch. All of them are rich in adenines and thymines but, surprisingly, these sequences differ from the wild-type SV40 AT stretch to such extent that at first sight they would seem unlikely to replicate. This is all the more impressive if we consider that another AT-rich sequence from the yeast TRP1 gene, which also carries a similar variation, cannot substitute for the SV40 AT stretch.

Adenine↗
PubMed1985

P1 plasmid replication: multiple functions of RepA protein at the origin.

Replication functions of a bacteriophage P1 miniplasmid are carried on a 1.2-kilobase pair (kb) segment that can be subdivided into a 245-base pair (bp) replication origin and a 959-bp region that encodes a protein required for replication (RepA). The origin region contains five 19-bp direct repeats. By using primer extension and gene-fusion assays, we mapped the promoter of the repA gene within the repeated sequences and showed that the promoter is repressed by RepA. Regulation of RepA synthesis is apparently achieved by the binding of RepA to the repeat sequences. This regulation might be a key step in the replication-control circuit, as we found that overproduction of RepA (from a foreign promoter) inhibits replication. Thus, in addition to being an autoregulated activator of replication, the protein also can have a negative regulatory role.

Bacteriophages↗
PubMed1980

Isolation of chromosomal origins of replication in yeast.

Origins of replication have been identified in the DNA of viruses, mitochondria, bacterial plasmids and the bacterial chromosome. However, origins of replication of eukaryote chromosomes have remained elusive because of the large size and sequence complexity of chromosomes and in particular for want of a suitable assay for their detection. Recent development of techniques for genetic transformation of yeast by autonomously replicating cytoplasmic plasmids now makes it possible to search for eukaryote origins in a manner analogous to that used for bacteria. Here we describe the construction and properties of a plasmid which contains no effective eukaryote replication origin and whose efficiency of replication in yeast is greatly enhanced by insertion of certain fragments of yeast chromosomal DNA. We believe these to contain replication origins, since yeast transformants are shown to contain copies of the transforming plasmids.

Base Sequence↗
PubMed2005

Cell cycle regulation of chromatin at an origin of DNA replication.

Selection and licensing of mammalian DNA replication origins may be regulated by epigenetic changes in chromatin structure. The Epstein-Barr virus (EBV) origin of plasmid replication (OriP) uses the cellular licensing machinery to regulate replication during latent infection of human cells. We found that the minimal replicator sequence of OriP, referred to as the dyad symmetry (DS), is flanked by nucleosomes. These nucleosomes were subject to cell cycle-dependent chromatin remodeling and histone modifications. Restriction enzyme accessibility assay indicated that the DS-bounded nucleosomes were remodeled in late G1. Remarkably, histone H3 acetylation of DS-bounded nucleosomes decreased during late G1, coinciding with nucleosome remodeling and MCM3 loading, and preceding the onset of DNA replication. The ATP-dependent chromatin-remodeling factor SNF2h was also recruited to DS in late G1, and formed a stable complex with HDAC2 at DS. siRNA depletion of SNF2h reduced G1-specific nucleosome remodeling, histone deacetylation, and MCM3 loading at DS. We conclude that an SNF2h-HDAC1/2 complex coordinates G1-specific chromatin remodeling and histone deacetylation with the DNA replication initiation process at OriP.

Adenosine Triphosphatases↗
PubMed2002

14-3-3sigma is a cruciform DNA binding protein and associates in vivo with origins of DNA replication.

A human cruciform binding protein (CBP) was previously shown to bind to cruciform DNA in a structure-specific manner and be a member of the 14-3-3 protein family. CBP had been found to contain the 14-3-3 isoforms beta, gamma, epsilon, and zeta. Here, we show by Western blot analysis that the CBP-cruciform DNA complex eluted from band-shift polyacrylamide gels also contains the 14-3-3sigma isoform, which is present in HeLa cell nuclear extracts. An antibody specific for the 14-3-3sigma isoform was able to interfere with the formation of the CBP-cruciform DNA complex. The effect of the same anti-14-3-3sigma antibody in the in vitro replication of p186, a plasmid containing the minimal replication origin of the monkey origin ors8, was also analyzed. Pre-incubation of total HeLa cell extracts with this antibody decreased p186 in vitro replication to approximately 30% of control levels, while non-specific antibodies had no effect. 14-3-3sigma was found to associate in vivo with the monkey origins of DNA replication ors8 and ors12 in a cell cycle-dependent manner, as assayed by a chromatin immunoprecipitation (ChIP) assay that involved formaldehyde cross-linking, followed by immunoprecipitation with anti-14-3-3sigma antibody and quantitative PCR. The association of 14-3-3sigma with the replication origins was maximal at the G(1)/S phase. The results indicate that 14-3-3sigma is an origin binding protein involved in the regulation of DNA replication via cruciform DNA binding.

14-3-3 Proteins↗
PubMed2006

The Sinorhizobium meliloti chromosomal origin of replication.

The predicted chromosomal origin of replication (oriC) from the alfalfa symbiont Sinorhizobium meliloti is shown to allow autonomous replication of a normally non-replicating plasmid within S. meliloti cells. This is the first chromosomal replication origin to be experimentally localized in the Rhizobiaceae and its location, adjacent to hemE, is the same as for oriC in Caulobacter crescentus, the only experimentally characterized alphaproteobacterial oriC. Using an electrophoretic mobility shift assay and purified S. meliloti DnaA replication initiation protein, binding sites for DnaA were mapped in the S. meliloti oriC region. Mutations in these sites eliminated autonomous replication. S. meliloti that expressed DnaA from a plasmid lac promoter was observed to form pleomorphic filamentous cells, suggesting that cell division was perturbed. Interestingly, this cell phenotype is reminiscent of differentiated bacteroids found inside plant cells in alfalfa root nodules.

Bacterial Proteins↗
PubMed1998

Skewed oligomers and origins of replication.

The putative origin of replication in prokaryotic genomes can be located by a new method that finds short oligomers whose orientation is preferentially skewed around the origin. The skewed oligomer method is shown to work for all bacterial genomes and one of three archaeal genomes sequences to date, confirming known or predicted origins in most cases and in three cases (H. pylori, M. thermoautotrophicum, and Synechocystis sp.), suggesting origins that were previously unknown. In many cases, the presence of conserved genes and nucleotide motifs confirms the predictions. An algorithm for finding these skewed seven-base and eight-base sequences is described, along with a method for combining evidence from multiple skewed oligomers to accurately locate the replication origin. Possible explanations for the phenomenon of skewed oligomers are discussed. Explanations are presented for why some bacterial genomes contain hundreds of highly skewed oligomers, whereas others contain only a handful.

Algorithms↗
PubMed1998

Concurrent replication and methylation at mammalian origins of replication.

Observations made with Escherichia coli have suggested that a lag between replication and methylation regulates initiation of replication. To address the question of whether a similar mechanism operates in mammalian cells, we have determined the temporal relationship between initiation of replication and methylation in mammalian cells both at a comprehensive level and at specific sites. First, newly synthesized DNA containing origins of replication was isolated from primate-transformed and primary cell lines (HeLa cells, primary human fibroblasts, African green monkey kidney fibroblasts [CV-1], and primary African green monkey kidney cells) by the nascent-strand extrusion method followed by sucrose gradient sedimentation. By a modified nearest-neighbor analysis, the levels of cytosine methylation residing in all four possible dinucleotide sequences of both nascent and genomic DNAs were determined. The levels of cytosine methylation observed in the nascent and genomic DNAs were equivalent, suggesting that DNA replication and methylation are concomitant events. Okazaki fragments were also demonstrated to be methylated, suggesting that the rapid kinetics of methylation is a feature of both the leading and the lagging strands of nascent DNA. However, in contrast to previous observations, neither nascent nor genomic DNA contained detectable levels of methylated cytosines at dinucleotide contexts other than CpG (i.e., CpA, CpC, and CpT are not methylated). The nearest-neighbor analysis also shows that cancer cell lines are hypermethylated in both nascent and genomic DNAs relative to the primary cell lines. The extent of methylation in nascent and genomic DNAs at specific sites was determined as well by bisulfite mapping of CpG sites at the lamin B2, c-myc, and beta-globin origins of replication. The methylation patterns of genomic and nascent clones are the same, confirming the hypothesis that methylation occurs concurrently with replication. Interestingly, the c-myc origin was found to be unmethylated in all clones tested. These results show that, like genes, different origins of replication exhibit different patterns of methylation. In summary, our results demonstrate tight coordination of DNA methylation and replication, which is consistent with recent observations showing that DNA methyltransferase is associated with proliferating cell nuclear antigen in the replication fork.

Animals↗