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PubMed1989

Negative control of plasmid R6K replication: possible role of intermolecular coupling of replication origins.

The gamma origin binding sites of the replication initiator pi protein, composed of seven 22-base-pair (bp) direct repeats and previously shown to be essential for replication of plasmid R6K, can also act as an inhibitor of R6K replication in Escherichia coli cells if provided in trans. Inhibition is dependent upon the ability of these repeats to bind the R6K-encoded pi protein but is not overcome by increasing the intracellular pi level. The insertion of a second repeat cluster in close proximity to the gamma origin also can markedly inhibit replication. The severity of this effect is dependent upon the position, orientation, and number of repeats present in the extra cluster. As few as six extra repeats can result in a completely nonfunctional gamma origin. However, this inactive gamma origin plasmid containing the six extra repeats is functional when placed in a strain that underproduces the wild-type pi protein or when placed in the presence of any of several copy-up mutant pi proteins. On the basis of these observations, we propose that the nucleoprotein structures formed by the binding of pi protein to the seven 22-bp direct repeats at the gamma origin are capable of coupling with each other in vivo and that replication initiation is prevented at such coupled origins. In support of this model of replication control, we demonstrate by electron microscopy analysis that the pi protein has the ability to associate two DNA molecules containing gamma origin sequences and also show that pi enhances the DNA ligase-catalyzed multimerization of a DNA fragment containing the gamma origin.

Bacterial Proteins↗
PubMed1997

A lesion in the DNA replication initiation factor Mcm10 induces pausing of elongation forks through chromosomal replication origins in Saccharomyces cerevisiae.

We describe a new minichromosome maintenance factor, Mcm10, and show that this essential protein is involved in the initiation of DNA replication in Saccharomyces cerevisiae. The mcm10 mutant has an autonomously replicating sequence-specific minichromosome maintenance defect and arrests at the nonpermissive temperature with dumbbell morphology and 2C DNA content. Mcm10 is a nuclear protein that physically interacts with several members of the MCM2-7 family of DNA replication initiation factors. Cloning and sequencing of the MCM10 gene show that it is identical to DNA43, a gene identified independently for its putative role in replicating DNA. Two-dimensional DNA gel analysis reveals that the mcm10-1 lesion causes a dramatic reduction in DNA replication initiation at chromosomal origins, including ORI1 and ORI121. Interestingly, the mcm10-1 lesion also causes replication forks to pause during elongation through these same loci. This novel phenotype suggests a unique role for the Mcm10 protein in the initiation of DNA synthesis at replication origins.

Alleles↗
PubMed1991

Recombination hotspots in bacteriophage T4 are dependent on replication origins.

Bacteriophage T4 recombination "hotspots" were first detected by the rescue of genetic markers from UV-irradiated phage particles. These hotspots have since been detected following treatments that yield other forms of DNA damage, and at least one is active in the absence of damage. The previous mapping of phage replication origins near the peaks of two recombination hotspots suggested that the origins cause the localized enhancement of recombination. Here we show that deletion of one origin eliminates the corresponding recombination hotspot, as judged by rescue of markers from UV-irradiated phage. Furthermore, insertion of either origin into a recombination "coldspot" enhances rescue of nearby markers. We conclude that these origins are necessary, and very likely sufficient, for the generation of recombination hotspots. We also show that the hotspots are active in the absence of both phage-encoded UvsY and host-encoded RecA proteins, suggesting that some of the stimulated recombination occurs by a synaptase-independent mechanism.

DNA Replication↗
PubMed1990

Structural modifications induced by the mtDBP-C protein in the replication origin of Xenopus laevis mitochondrial DNA.

The structure of the non-coding region of Xenopus laevis mitochondrial DNA has been studied by electron microscopy analysis of DNA molecules end-labelled with streptavidin-ferritin. We have shown that the effect of a protein modifying the shape of the DNA double-helix can be studied and precisely located by this method. It was found that the non-coding region contains curved segments and that the mitochondrial protein mtDBP-C preferentially enhances the curvature of the promoters-replication origin region.

Animals↗
PubMed1989

The 18-kD protein that binds to the chloroplast DNA replicative origin is an iron-sulfur protein related to a subunit of NADH dehydrogenase.

From a high-salt extract of the purified thylakoid membrane, an 18-kD protein was detected. This protein was translated by the chloroplast ribosomes and could form a stable DNA-protein complex with a cloned chloroplast DNA replicative origin [Nie, Z.Q., Chang, D.Y., and Wu, M. (1987) Mol. Gen. Genet. 209, 265-269]. In this paper, the 18-kD protein is linked to frxB, a chloroplast-encoded, ferredoxin-type, iron-sulfur protein, by N-terminal microsequencing of the purified protein and computer analysis. The identification is further supported empirically by the fact that the electron paramagnetic resonance spectra of the protein indicate the presence of iron-sulfur clusters. A polyclonal antibody raised against a synthetic pentadecameric peptide with amino acid sequence corresponds to the highly conserved region of the frxB protein and reacts strongly and specifically with the 18-kD protein band in protein gel blot analyses. The 18-kD iron-sulfur protein is found to be related to a subunit of the respiratory chain NADH dehydrogenase by its cross-reaction with a polyclonal antibody raised against highly purified NADH-ubiquinone oxidoreductase, a key enzyme of the respiratory chain. These data are consistent with chlororespiration, and, thus, possible implication of chlororespiration in regulating the initiation of chloroplast DNA replication is discussed.

Amino Acid Sequence↗
PubMed2000

Stepwise assembly of initiation proteins at budding yeast replication origins in vitro.

The initiation of DNA replication in the budding yeast Saccharomyces cerevisiae occurs in two sequential and mutually exclusive steps. Prereplicative complexes (pre-RCs) containing origin recognition complex (ORC), Cdc6p, and the MCM2-7 proteins assemble only under conditions of low cyclin-dependent kinase (Cdk) activity during G(1), whereas origin activation is driven by the increase in Cdk activity at the end of G(1). As a first step toward the reconstitution of this two-step process in vitro, we describe a system in which extracts prepared from G(1)-arrested cells promote sequential assembly of ORC, Cdc6p, and MCM2-7 proteins onto exogenously added origin-containing DNA. This reaction requires an intact ARS consensus sequence and requires ATP for two distinct steps. Extracts from cells arrested in mitosis also can support the binding of ORC but are unable to load either Cdc6p or MCM2-7 proteins. This system should be useful for studying the mechanism and regulation of pre-RC assembly.

Adenosine Triphosphate↗
PubMed1995

Replication efficiency of bovine papillomavirus type 1 DNA depends on cis-acting sequences distinct from the replication origin.

The viral elements required for the initiation of replication of bovine papillomavirus type 1 DNA include the origin region and two trans-acting factors, the E1 and E2 proteins. We now report that the replication efficiency of a DNA molecule which contains these three elements is modulated by other viral sequences. By measuring the extent of replication of deleted viral genomes in transfected mouse cells, we identified sequences required for maximal efficiency. Addition of these sequences to a construct carrying only the minimal origin region increased its replication. Among these cis-active elements, we identified a 69-bp fragment (nucleotides 4921 to 4990) which contains at least two binding sites for cellular proteins. One of them is the murine protein termed CDEBP, which recognizes the octameric motif ATCACGTG, identical to the yeast CDEI element. Either deletions affecting this CDEI box or a point mutation which impairs binding of CDEBP markedly decreased the extent of viral DNA replication. They had no detectable effect on viral transcription.

Animals↗
PubMed1990

The Bacillus subtilis small cytoplasmic RNA gene and 'dnaX' map near the chromosomal replication origin.

The Bacillus subtilis small cytoplasmic RNA (scRNA) has an important, although not yet defined function in protein biosynthesis. Here we describe the mapping of the single copy scRNA gene and the flanking homolog to dnaZX of Escherichia coli, termed 'dnaX'. The scRNA gene region of a B. subtilis wild-type strain was marked with a cat gene and mapped by scoring chromosomal cotransformation rates of various mutant strains to chloramphenicol resistance and loss of the mutant phenotypes, respectively. This analysis, together with an EcoRI map comparison, places the scRNA gene and dnaX in the vicinity of recM near the replication origin region of B. subtilis.

Bacillus subtilis↗
PubMed1978

Bacteriophage lambda carrying the Escherichia coli chromosomal region of the replication origin.

A transducing phage lambdaasn was isolated. The late gene region of its genome was found to have been substituted by an Escherichia coli chromosomal segment containing the genes bgIR, bgIC, glmS, uncA, and asn. Restriction endonuclease cleavage mapping and electron microscopic analysis of the lambdaasn DNA revealed that the size of the bacterial segment is approximately 1.75 X 10(7) daltons, corresponding to about 26.4 kilobases. The circular DNA of lambdaasn was digested with restriction endonuclease EcoRI, diluted, and sealed with DNA ligase. When the reaction mixture was used to transform a recipient E. coli strain, a small plasmid of about 1 X 10(7) daltons (named pMCR115) was obtained. Restriction endonuclease cleavage mapping of pMCR115 and other evidence suggested that it contained the replication origin (oriC) of the E. coli chromosome.

Chromosomes, Bacterial↗
PubMed1993

Structural organization of the maxicircle variable region of Trypanosoma brucei: identification of potential replication origins and topoisomerase II binding sites.

The maxicircle of the parasitic protozoan Trypanosoma brucei, one component of the mitochondrial genome, has size differences among isolates that localize to the variable region (VR) between the ND5 and 12S rRNA genes. We present here the nucleotide sequence of this entire region, thus completing the sequence of the maxicircle genome. We also find heterogeneously sized transcripts from throughout most of the VR. The VR has three distinct sections, each with characteristic repeated sequences. The repeated sequences in two sections are short and highly reiterated; the intraspecies size variation occurs within this region. The third section contains non-repetitive sequences and a large duplication immediately upstream of the 12S rRNA gene. Two repeat units within section I contain a sequence that has homology to the DNA replication origin of minicircles. This region also contains sequences with homology to topoisomerase II binding and cleavage sites. These findings suggest a role for the VR in DNA replication of the maxicircle.

Animals↗
PubMed1981

Structure and function of the region of the replication origin of the Bacillus subtilis chromosome. I. Isolation and characterization of plasmids containing the origin region.

A BamHI restriction endonuclease fragment, B7, which is replicated first among all other fragments derived from the Bacillus subtilis chromosome, was cloned in Escherichia coli using as vector a hybrid plasmid pMS102 that can replicate both in E. coli and B. subtilis. Digestion of pMS102 with BamHI produced two fragments and the smaller one was replaced by the B7 fragment. The cloned plasmid pMS102'-B7 exhibited some peculiar properties that were not observed with plasmids containing other fragments from the B. subtilis chromosome. (1) E. coli cells harboring this plasmid stuck to each other and to glass. This property was more apparent when cells were grown in poor media. (2) E. coli cells tended to lose the plasmid spontaneously when they were grown without the selective pressure favorable to the plasmid. (3) The frequency of transformation of B. subtilis by pMS102'-B7 was less than 1/1,000 of that by the vector plasmid pMS102'. The number of copies of pMS102'-B7 present in the transformants was also markedly reduced, although the pUB110 origin of replication on the vector was intact and should be functional in B. subtilis. This inhibitory effect of the B7 fragment on plasmid replication was confirmed more directly by developing a semi in vitro replication system using protoplasts. Both in E. coli and B. subtilis the B7 fragment affected replication of its own molecule but not that of the coexisting plasmid with an identical replication system. The implication of the function of the B7 fragment in the initiation of the B. subtilis chromosome will be discussed.

Bacillus subtilis↗
PubMed1999

Radiation down-regulates replication origin activity throughout the S phase in mammalian cells.

An asynchronous culture of mammalian cells responds acutely to ionizing radiation by inhibiting the overall rate of DNA replication by approximately 50% for a period of several hours, presumably to allow time to repair DNA damage. At low and moderate doses, this S phase damage-sensing (SDS) pathway appears to function primarily at the level of individual origins of replication, with only a modest inhibition of chain elongation per se. We have shown previously that the majority of the inhibition observed in an asynchronous culture can be accounted for by late G1cells that were within 2-3 h of entering the S period at the time of irradiation and which then fail to do so. A much smaller effect was observed on the overall rate of replication in cells that had already entered the S phase. This raised the question whether origins of replication that are activated within S phase per se are inhibited in response to ionizing radiation. Here we have used a two-dimensional gel replicon mapping strategy to show that cells with an intact SDS pathway completely down-regulate initiation in both early- and late-firing rDNA origins in human cells. We also show that initiation in mid- or late-firing rDNA origins is not inhibited in cells from patients with ataxia telangiectasia, confirming the suggestion that these individuals lack the SDS pathway.

Animals↗
PubMed1985

Binding and bending of the lambda replication origin by the phage O protein.

We have characterized the binding of lambda phage replication initiation protein O to the phage origin of replication. The minimal DNA segment required for O binding is the single iteron, a 19-bp sequence of hyphenated dyad symmetry that is repeated with variations four times in the origin. The isolated amino terminus of O protein is also sufficient to bind DNA. Electrophoretic studies show that the amino terminus of O protein induces bending of a single iteron. The DNA-protein interaction was characterized by ethylation interference, dimethyl sulfate protection and neocarzinostatin footprinting. Points of DNA-protein contact are largely concentrated in two areas symmetrically disposed with respect to the dyad symmetry of the iteron. This suggests the protein interacts as a dimer with half sites in the DNA. However, a few non-symmetrical contacts are found, indicating that O protein may distort the helix. This may correlate with the bending effects demonstrated electrophoretically. Cylindrical DNA projections were used to model O protein binding to the lambda origin and compare it with the lambda repressor-operator interaction. Whereas bound repressor nearly encircles the DNA in the major groove, O protein leaves the major groove on the opposite side exposed.

Alkylation↗
PubMed1984

Replication origins and a sequence involved in coordinate induction of the immediate-early gene family are conserved in an intergenic region of herpes simplex virus.

We have determined the structure of the 5' portion of herpes simplex virus type 2 (HSF-2) immediate-early (IE) mRNA-3 and have obtained the DNA sequence specifying the N terminus of its encoded polypeptide, Vmw182, its untranslated leader and the intergenic region between IEmRNAs-3 & 4/5. Comparison of the HSV-2 intergenic sequences with the HSV-1 equivalent region identifies several conserved regions: (1) an AT-rich element with core consensus TAATGARAT which is likely to be the 'activator' sequence through which coordinate induction of the IE gene family is mediated. (2) GC-rich and GA-rich tracts, found in a wide variety of eukaryotic promoters, which vary in position and orientation between HSV-2 and HSV-1 and which represent modulators of transcription. (3) TATA homologies present 15-25 base pairs (bp) upstream of mRNA 5' termini. (4) a 137bp direct repeat in HSV-2 which contains sequence almost identical to the HSV-1 replication origin.

Amino Acid Sequence↗
PubMed1980

Mapping of SV40 DNA replication origin region binding sites for the SV40 T antigen by protection against exonuclease III digestion.

Incubation of 32P-5' end-labeled DNA fragments of less than 500 bp with excess amounts of the 3' leads to 5', double strand-dependent nuclease Exonuclease III generally results in single-stranded products of slightly more than half the size of the uncleaved substrate. When such restriction fragments of known size and sequence containing the lac operator were incubated with purified lac repressor, Exonuclease III cleavage was blocked at the 3' borders of the operator on each strand. It was possible to define the DNA sequence between the two boundaries of repressor-mediated exonuclease blockade by electrophoresing the single-stranded, protected products in urea-containing polyacrylamide gels in parallel with a dimethylsulfate modification-cleavage digest of the end-labeled, uncleaved substrate. The same approach was applied to an analysis of sites of large SV40 T antigen protection in the vicinity of the origin of SV40 DNA replication. Three discrete boundaries of apparent protection were observed--one on the "late" side of the origin and two on the "early" side. These sequences may constitute the 3' borders of discrete T antigen-binding sites in the origin region. Alternatively, one or more of these blockade points may signify regions of the genome which undergo conformational changes resulting in Exonuclease III resistance due to vicinal T antigen binding.

Antigens, Viral↗
PubMed1985

Properties of some monkey DNA sequences obtained by a procedure that enriches for DNA replication origins.

Twelve clones of monkey DNA obtained by a procedure that enriches 10(3)- to 10(4)-fold for nascent sequences activated early in S phase (G. Kaufmann, M. Zannis-Hadjopoulos, and R. G. Martin, Mol. Cell. Biol. 5:721-727, 1985) have been examined. Only 2 of the 12 ors sequences (origin-enriched sequences) are unique (ors1 and ors8). Three contain the highly reiterated Alu family (ors3, ors9, and ors11). One contains the highly reiterated alpha-satellite family (ors12), but none contain the Kpn family. Those remaining contain middle repetitive sequences. Two examples of the same middle repetitive sequence were found (ors2 and ors6). Three of the middle repetitive sequences (the ors2-ors6 pair, ors5, and ors10) are moderately dispersed; one (ors4) is highly dispersed. The last, ors7, has been mapped to the bona fide replication origin of the D loop of mitochondrial DNA. Of the nine ors sequences tested, half possess snapback (intrachain reannealing) properties.

Animals↗
PubMedDate not supplied

An in vitro method generating base substitutions in preselected regions of plasmid DNA: application to structural analysis of the replication origin of the Escherichia coli K-12 chromosome.

A method for introducing base substitutions in defined regions of plasmid DNA has been developed. In principle, a circular heteroduplex DNA containing a gap is constructed by annealing of two kinds of linear molecules derived from the same plasmid: One is the molecule shortened either by exonucleolytic digestion from the termini generated at a restriction site or by removal of a region flanked by two restriction sites, and the other the full-length molecule linearized at a different site. The deleted region in the shorter linear molecule becomes a single-stranded gap in the circular heteroduplex DNA. The heteroduplex is then treated with sodium bisulfite that converts specifically cytosine residues to uracil residues in single-stranded regions. After filling in the gap by repair synthesis, transformation is carried out to isolate mutant plasmids. Since two kinds of circular heteroduplexes are formed by annealing in which the sequences in the gaps are complementary to each other, mutagenesis of both strands can be accomplished in one experiment. This method was applied to construction of mutants with base substitutions in the replication origin region (oriC) of the Escherichia coli K-12 chromosome which had previously been cloned in colicin E1 plasmid vectors, and various mutants in defined regions of oriC were successfully isolated at high efficiencies. Analysis of these mutants provided evidence that oriC contains special regions, designated spacers, which separate neighboring important sequences specifying interactions with initiation factors for DNA replication at precise distances.

Base Sequence↗
PubMed1993

Recombinant human hepatitis B virus reverse transcriptase is active in the absence of the nucleocapsid or the viral replication origin, DR1.

The double-stranded DNA genome of hepatitis B virus (HBV) is reverse transcribed from the viral pregenome RNA template by a virally encoded reverse transcriptase enzyme (RT) that possesses both priming and elongation activities. Prior efforts have failed to express an active form of HBV RT outside the nucleocapsid in animal cells or to release it from viral nucleocapsids, thus restricting the characterization of this important enzyme. Here, we have engineered epitope-tagged HBV RT proteins and expressed them in Xenopus oocytes via a synthetic RT mRNA which does not include the viral capsid protein or the known initiation site for viral DNA synthesis, DR1. We demonstrate the production of an immunoprecipitable 96-kDa HBV RT protein and show, using a simple in vitro RT assay, that oocyte lysates containing this protein possess an activity that (i) catalyzes an RNA-dependent deoxynucleotide triphosphate polymerization reaction by using an as-yet-unidentified RNA template and (ii) is sensitive to the RT inhibitors actinomycin D and phosphonoformate. Experiments with the chain terminator ddATP suggest that a significant amount of chain elongation occurs in our in vitro reaction. Electrophoretic analysis reveals a heterogeneous array of RT reaction products with sizes ranging from about 100 bases to far larger than that of the input RT mRNA. These products appear to contain covalently bound protein, consistent with the notion that the RT protein may have primed their synthesis. We conclude that HBV RT activity can be uncoupled from both the nucleocapsid and the replication origin, DR1. Our results raise the possibility that unless HBV employs novel mechanisms to regulate its constitutively active RT, cellular RNAs may be reverse transcribed during HBV infection, with potential implications for the development of HBV-related liver cancer. The use of the oocyte system should facilitate studies of HBV RT, including the development of HBV RT inhibitors for antiviral therapy.

Amino Acid Sequence↗