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PubMed1991

Identification of two distinct regions within the adenovirus minimal origin of replication that are required for adenovirus type 4 DNA replication in vitro.

The adenovirus type 4 origins of replication are located at each end of the linear, protein-linked viral DNA molecule and consist of the terminal 18 bp of the viral genome. The sequence of the first 8 bp of the viral genome varies among different adenovirus serotypes, but the sequence from bp 9 to 18 is conserved in all human serotypes, suggesting that it may be of critical importance to origin function. Using an in vitro system in which purified fractions or crude extracts of adenovirus type 4-infected HeLa cells can support initiation and elongation on linearized plasmid templates containing cloned origin sequences, we examined the effect of single base changes in positions 9 to 18 of the adenovirus origin on DNA replication in vitro. Changes in positions 12 to 16 have little effect, whereas alterations at positions 9, 10, 11, 17, and 18 all reduce the efficiency of initiation of DNA replication by between 50 and 90%. Our results show that the region from bp 9 to 18 contains two sets of bases essential for DNA replication which are separated by 5 bp in which single base changes can be accommodated. The likely role of the region from bp 9 to 18 as containing the recognition sequence for a DNA-protein interaction essential for viral DNA replication is discussed.

Adenoviruses, Human↗

PubMed1994

Mapping an initiation region of DNA replication at a single-copy chromosomal locus in Drosophila melanogaster cells by two-dimensional gel methods and PCR-mediated nascent-strand analysis: multiple replication origins in a broad zone.

We have mapped an initiation region of DNA replication at a single-copy chromosomal locus in exponentially proliferating Drosophila tissue culture cells, using two-dimensional (2D) gel replicon mapping methods and PCR-mediated analysis of nascent strands. The initiation region was first localized downstream of the DNA polymerase alpha gene by determining direction of replication forks with the neutral/alkaline 2D gel method. Distribution of replication origins in the initiation region was further analyzed by using two types of 2D gel methods (neutral/neutral and neutral/alkaline) and PCR-mediated nascent-strand analysis. Results obtained by three independent methods were essentially consistent with each other and indicated that multiple replication origins are distributed in a broad zone of approximately 10 kb. The nucleotide sequence of an approximately 20-kb region that encompasses the initiation region was determined and searched for sequence elements potentially related to function of replication origins.

Animals↗

PubMed1987

Nuclei act as independent and integrated units of replication in a Xenopus cell-free DNA replication system.

We have used a novel approach to investigate the control of initiation of replication of sperm nuclei in a Xenopus cell-free extract. Nascent DNA was labelled with biotin by supplementing the extract with biotin-11-dUTP, and isolated nuclei were then probed with fluorescein-conjugated streptavidin. Flow cytometry was used to measure the biotin content of individual nuclei and their total DNA content. This showed that incorporation of the biotinylated precursor increases linearly with DNA content. Haploid sperm nuclei replicate fully to reach the diploid DNA content over 2-6 h in the extract. Synthesis stops once the diploid DNA content is reached. Different nuclei enter S phase at different times over greater than 1.5 h, although they share the same cytoplasmic environment. Nuclei reach their maximum rates of synthesis soon after entry into S phase and some replicate fully in less than 0.5 h, resembling the rates of replication observed in the intact egg. These results indicate that initiations are coordinated within each nucleus such that the nucleus is the fundamental unit of replication in the cell-free system.

Animals↗

PubMed1987

Start sites for bidirectional in vitro DNA replication inside the replication origin, oriC, of Escherichia coli.

In vitro replication of mini-chromosomes in the absence of DNA ligase activity resulted in replication products with single-strand breaks at specific sites. The occurrence of these nicks was coupled to an active replication process, therefore we expect them to represent start sites for DNA replication. Two positions within oriC for each of the two leading strands of bidirectional replication were found. Within each position are one or two start sites. Counterclockwise synthesis started at positions 194/199 and 265/272, clockwise synthesis at positions 209/219 and 254. The start positions are located close to DnaA protein binding sites. A model for initiation accommodating this observation is discussed.

Chromosomes, Bacterial↗

PubMed1995

Termination of DNA replication in vitro: requirement for stereospecific interaction between two dimers of the replication terminator protein of Bacillus subtilis and with the terminator site to elicit polar contrahelicase and fork impedance.

The termination of DNA replication at a sequence-specific replication terminus in Bacillus subtilis is catalyzed by a dimeric replication terminator protein (RTP) of subunit mol. wt 14,500. RTP has become an attractive protein with which to study the molecular mechanism of termination because its crystal structure has now been solved and the previous lack of an in vitro replication system has been largely overcome by our discovery that the protein terminates replication in vivo and in vitro in the well-studied Gram-negative Escherichia coli system. We have exploited the surrogate in vitro system to show that RTP acts as a polar contrahelicase to DnaB helicase of E. coli only when two RTP dimers are bound co-operatively to overlapping core and auxiliary sequences comprising the terminus. A core sequence by itself binds one dimer of RTP, but elicits no contrahelicase activity. Binding of two RTP dimers to a tandem head-to-tail core repeat also elicits no contrahelicase activity, thus suggesting that a specific stereochemical interaction between two RTP dimers and with the terminator site is essential for termination. RTP blocks unwinding of DNA substrates containing heteroduplex regions that include the terminus and are in the size range of approximately 50 to > 1000 bp in length. Thus, the protein blocks authentic helicase-catalyzed unwinding rather than just the translocation of the helicase on DNA.

Bacillus subtilis↗

PubMed1995

Deletion analysis of minimal sequence requirements for autonomous replication of ors8, a monkey early-replicating DNA sequence.

We have generated a panel of deletion mutants of ors8 (483 bp), a mammalian autonomously replicating DNA sequence, previously isolated by extrusion of nascent monkey (CV-1) DNA from replication bubbles active at the onset of S phase. The deletion mutants were tested for replication function by the DpnI resistance assay, in vivo, after transfection into HeLa cells, and in vitro. An internal fragment of 186-bp that is required for autonomous replication function of ors8 was identified. This fragment, when subcloned into pBR322 and similarly tested, was capable of autonomous replication in vivo and in vitro. The 186-bp fragment contains several repeated sequence motifs, such as the ATTA and ATTTAT motifs, occurring three and five times, respectively, the sequences TAGG and TAGA, occurring three and seven times, respectively, two 5'-ATT-3' repeats, a 44-bp imperfect inverted repeat (IR) sequence, and an imperfect consensus binding element for the transcription factor Oct-1. A measurable sequence-directed DNA curvature was also detected, coinciding with the AT-rich regions of the 186-bp fragment.

Animals↗

PubMed1979

Maturation of replicating simian virus 40 DNA molecules in isolated nuclei by continued bidirectional replication to the normal termination region.

Mature SV40 DNA synthesized for different periods of time either in isolated nuclei or in intact cells was highly purified and then digested with restriction endonucleases in order to relate the time of synthesis of newly replicated viral DNA to its location in the genome. Replication in nuclei supplemented with a cytosol fraction from uninfected cells was a faithful continuation of the bidirectional process observed in intact cells, but did not exhibit significant initiation of new replicons. SV40 DNA replication in cells at 37 degrees C proceeded at about 145 nucleotides/min per replication fork. In the absence of cytosol, when DNA synthesis was limited and joining of Okazaki fragments was retarded, bidirectional SV40 DNA replication continued into the normal region where separation yeilded circular duplex DNA molecules containing one or more interruptions in the nascent DNA strands. In the presence of cytosol, this type of viral DNA was shown to be a precursor of covalently closed, superhelical SV40 DNA, the mature from of viral DNA.

Animals↗

PubMed1986

Regulation of lambda dv plasmid DNA replication. A quantitative model for control of plasmid lambda dv replication in the bacterial cell division cycle.

A quantitative model for the regulation of replication of plasmid lambda dv in the Escherichia coli cell division cycle has been developed. The regulatory processes include the interactions of cro repressor proteins with the rightward operator DNA sites, the transcriptional activation of the lambda dv replication origin, and the interaction of initiation proteins with activated origins to form functional replication initiation complexes. A statistical thermodynamic model was used to predict probable configurations of the regulatory processes in a single growing cell. These probabilities were coupled by a kinetic model to the events of the cell cycle such as initiation of mRNA transcription and protein translation and the initiation of plasmid DNA replication. Parameter values were chosen so that the simulated values for plasmid copy number and repressor and initiator protein concentrations of the model agreed with experimentally determined estimates. Simulated deviations from regular segregation of the various components at cell division, such as plasmid copies and free and bound repressor proteins, suggest that lambda dv replication control responds only slowly to these perturbations. The consequence of this slow response to perturbations, which are expected at a random frequency, was simulated for a population of lambda dv-containing cells in a growing culture. This simulation predicts instability of inheritance of lambda dv plasmids in the population, despite the very high plasmid copy number, in agreement with experimental observation.

Bacteriophage lambda↗

PubMed1992

Comparison of replicative and non-replicative chromatin assembly pathways in HeLa cell extracts.

It has been reported that chromatin assembly in mammalian cell extracts depends exclusively or preferentially on ongoing DNA replication (Stillman, B. (1986) Cell 45, 555-565). More recently, this view has been challenged demonstrating that, in the same extracts, chromatin can also be formed efficiently in the absence of DNA replication (Gruss et al. (1990) EMBO J. 9, 2911-2922). The experiments, described in this communication, were performed to resolve this apparent contradiction. We found that there are at least two distinct in vitro pathways for chromatin assembly in HeLa cell extracts. The replicative pathway requires a nuclear protein, most likely identical with the chromatin assembly factor, described by Stillman (1986, Cell 45, 555-565), and the free soluble histones present in the cytosol of S phase cells. In contrast, a non-replicative pathway was identified that depends on isolated nuclear histones. As one component of the non-replicative assembly pathway we identified a cytosolic factor that was purified to apparent homogeneity and shown to be an acidic 50 kDa polypeptide. The isolated cytosolic 50 kDa protein efficiently promoted nucleosome assembly as demonstrated by one- and two-dimensional gel electrophoresis of in vitro packaged plasmid DNA.

Antigens, Polyomavirus Transforming↗

PubMed1979

Inceptor and origin of DNA replication in lambdoid coliphages. II. The lambda DNA maximal replication system.

In pBR313-lambda dv hydrid plasmids a second system for initiation of DNA replication has been detected in lambdoid replicator DNAs (in the absence of the p0 promoter). The "maximal" (or "maxi") initiation system depends on the origin of replication (ori) sequence, in conjuction with the "inceptor" (ice) element located in the lambdoid cII genes. Only leftward, but not bidirectional, primer RNA synthesis seems to be initiated at ori in its newly defined boundaries, and it appears to be catalysed by dnaG-coded primase. Only if transcriptionally activated, will ori effectively initiate lambda specific, O and P-dependent "maximal" hybrid-plasmid replication. In addition, it will repress a complete lambda "minimal" initiation system in cis, i.e., if present on the same plasmid molecule. This newly discovered repressive activity of the ori system depends on only three factors: an intact left section of ori, the O product, and transcriptional activation of ori (rightward or leftward). A repressed minimal initiation system will regain its activity as soon as a segment carrying either part of the O gene or a promoter for transcriptional activation is delected from such a plasmid which was combining both the "mini" and "maxi" systems of lambda replication.

Bacteriophage lambda↗

PubMed1991

Fidelity of mammalian DNA replication and replicative DNA polymerases.

Current models suggest that two or more DNA polymerases may be required for high-fidelity semiconservative DNA replication in eukaryotic cells. In the present study, we directly compare the fidelity of SV40 origin-dependent DNA replication in human cell extracts to the fidelity of mammalian DNA polymerases alpha, delta, and epsilon using lacZ alpha of M13mp2 as a reporter gene. Their fidelity, in decreasing order, is replication greater than or equal to pol epsilon greater than pol delta greater than pol alpha. DNA sequence analysis of mutants derived from extract reactions suggests that replication is accurate when considering single-base substitutions, single-base frameshifts, and larger deletions. The exonuclease-containing calf thymus DNA polymerase epsilon is also highly accurate. When high concentrations of deoxynucleoside triphosphates and deoxyguanosine monophosphate are included in the pol epsilon reaction, both base substitution and frameshift error rates increase. This response suggests that exonucleolytic proofreading contributes to the high base substitution and frameshift fidelity. Exonuclease-containing calf thymus DNA polymerase delta, which requires proliferating cell nuclear antigen for efficient synthesis, is significantly less accurate than pol epsilon. In contrast to pol epsilon, pol delta generates errors during synthesis at a relatively modest concentration of deoxynucleoside triphosphates (100 microM), and the error rate did not increase upon addition of adenosine monophosphate. Thus, we are as yet unable to demonstrate that exonucleolytic proofreading contributes to accuracy during synthesis by DNA polymerase delta. The four-subunit DNA polymerase alpha-primase complex from both HeLa cells and calf thymus is the least accurate replicative polymerase. Fidelity is similar whether the enzyme is assayed immediately after purification or after being stored frozen.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed1974

Mechanism of DNA replication in Drosophila chromosomes: structure of replication forks and evidence for bidirectionality.

The replicating chromosomal DNA in Drosophila melanogaster cleavage nuclei has been visualized in the electron microscope as a serial array of closely spaced replicated regions created by pairs of diverging replication forks. The fine structure of the forks is very similar to that observed for the replication forks of bidirectionally replicating bacteriophage DNAs. However, the mean length of the single-stranded gaps in Drosophila forks is less than 200 nucleotide residues, much shorter than the gaps in phage forks. This difference in gap length corresponds to the observed difference in the size of Okazaki fragments from Drosophila and phage.

Animals↗

PubMed1974

Replication of colicin E1 plasmid DNA in cell extracts. Origin and direction of replication.

The structures of molecules of colicin E1 plasmid DNA that were in the process of being replicated in vitro were examined electron microscopically. Circular molecules containing a loop of approximately 7% of the length of the molecules were the major class of replicating molecules. The location of the loop was determined by treating the molecules with restriction endonuclease EcoR1, which introduced one unique double-strand break in the colicin E1 plasmid DNA molecules. The loops had a specific location with two branch points at approximately 17 and 24% of the molecular length from the endonuclease-sensitive site. Molecules with a larger loop were observed with a preparation labeled with 5-bromodeoxyuridine and enriched for these molecules. One of the branch points in these molecules was located at approximately 17% of the molecular length from the endonuclease-sensitive site independent of the size of the loops. These results indicate that the origin of replication of the plasmid DNA is located within the small loop and the replication proceeds unidirectionally. The molecules that had completed a round of replication had the monomeric twisted circular structure.

Bromodeoxyuridine↗

PubMed1975

Bidirectional replication of plasmid R6K DNA in Escherichia coli; correspondence between origin of replication and position of single-strand break in relaxed complex.

Replicating molecules of plasmid R6K DNA have been purified as covalently closed circular DNA forms and analyzed in the electron microscopy after cleavage with the EcoRI restriction endonuclease. It has been determined that in most cases replication proceeds bidirectionally from an origin whose position is indistinguishable from the site of the single-strand break (nick) in the open circular DNA form of the relaxation complex of R6K DNA and protein. Evidence is presented for the existence of a unique replication terminus asymmetrically placed approximately 20% of genome size from the origin. The positions of the replication forks in a majority of the molecules indicate that replication proceeds sequentially from the fixed origin first in one direction to the terminus and then progresses from the origin in the other direction.

DNA Replication↗

PubMed1977

Role of DNA gyrase in phiX replicative-form replication in vitro.

Preparations containing DNA gyrase activity Gellert, M., Mizuchi, K., O'Dea, M.H. & Nash, H.A. (1976) Proc. Natl. Acad. Sci. USA 73, 3872-3876] have been extensively purified from Escherichia coli. Such fractions, in the presence of ATP and Mg2+, catalyze supertwisting of relaxed circular double-stranded DNA replicative forms of a number of DNAs that results in the formation of superhelical replicative forms. Relaxed phiX174 replicative form (phiX RFIV) is not attacked by the A protein endonuclease coded for by the phiX DNA genome. After exposure to preparations of DNA gyrase, the relaxed phiX174 replicative form is converted to phiX RFI which can then be attacked by the phiX gene A protein and participate in replication of duplex phiX DNA.

Adenosine Triphosphate↗

PubMed1992

Initiation of phi 29 DNA replication occurs at the second 3' nucleotide of the linear template: a sliding-back mechanism for protein-primed DNA replication.

Bacteriophage phi 29 DNA replication is initiated when a molecule of dAMP is covalently linked to a free molecule of the terminal protein, in a reaction catalyzed by the viral DNA polymerase. We demonstrate that single-stranded DNA molecules are active templates for the protein-primed initiation reaction and can be replicated by phi 29 DNA polymerase. Using synthetic oligonucleotides, we carried out a mutational analysis of the phi 29 DNA right end to evaluate the effect of nucleotide changes at the replication origin and to determine the precise initiation site. The results indicate that (i) there are no strict sequence requirements for protein-primed initiation on single-stranded DNA; (ii) initiation of replication occurs opposite the second nucleotide at the 3' end of the template; (iii) a terminal repetition of at least two nucleotides is required to efficiently elongate the initiation complex; and (iv) all the nucleotides of the template, including the 3' terminal one, are replicated. A sliding-back model is proposed in which a special transition step from initiation to elongation can account for these results. The possible implication of this mechanism for the fidelity of the initiation reaction is discussed. Since all the terminal protein-containing genomes have some sequence reiteration at the DNA ends, this proposed sliding-back model could be extrapolable to other systems that use proteins as primers.

Bacillus Phages↗

PubMed1992

CDC46/MCM5, a yeast protein whose subcellular localization is cell cycle-regulated, is involved in DNA replication at autonomously replicating sequences.

Saccharomyces cerevisiae cells containing mutations in the cell-division-cycle gene CDC46 arrest with a large bud and a single nucleus with unreplicated DNA at the non-permissive temperature. This G1/S arrest, together with the increased rates of mitotic chromosome loss and recombination phenotype, suggests that these mutants are defective in DNA replication. The subcellular localization of the CDC46 protein changes with the cell cycle; it is nuclear between the end of M phase and the G1/S transition but is cytoplasmic in other phases of the cell cycle. Here we show that CDC46 is identical to MCM5, based on complementation analysis of the mcm5-1 and cdc46-1 alleles, complementation of the minichromosome maintenance defect of mcm5-1 by CDC46, and the genetic linkage of these two genes. Like mcm5-1, cdc46-1 and cdc46-5 also show a minichromosome maintenance defect thought to be associated with DNA replication initiation at autonomously replicating sequences. Taken together, these observations suggest that CDC46/MCM5 acts during a very narrow window at the G1/S transition or the beginning of S phase by virtue of its nuclear localization to effect the initiation of DNA replication at autonomously replicating sequences.

Amino Acid Sequence↗

PubMed1982

DNA replication in Physarum polycephalum: electron microscopic analysis of patterns of DNA replication in the presence of cycloheximide.

DNA from synchronously replicating nuclei of Physarum polycephalum was studied electron microscopically after 15, 30, 60, and 90 or 120 min of replication in the presence or absence of the protein synthesis inhibitor cycloheximide. The replication-loop size-distribution showed that replication fork progression is severely retarded in the presence of cycloheximide. Analysis of replication-loop frequency showed a similar pattern in control and cyclo-heximide-treated samples, with an increase from 15 to 30 and 60 min. This suggests, surprisingly, that initiations of new replicons either may not be inhibited by cycloheximide or, alternatively, that all initiations have already taken place at the very start of S-phase. The latter conclusion is favored in the light of previous results in our laboratory, discussed here.

Cycloheximide↗