PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “replication”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 55 records · Page 3Linked to original sources
PubMed1992

Patterns of DNA replication in Drosophila polytene nuclei replicating in Xenopus egg and oocyte extracts.

We have used Xenopus laevis cell-free extracts to study patterns of DNA replication in polytene nuclei isolated from salivary glands of Drosophila melanogaster 3rd instar larvae. Replication was visualized by supplementation with biotin-dUTP so that nascent DNA became labelled, thus allowing detection with fluorescein or Texas-Red-conjugated streptavidin. Biotin incorporation was dependent on incubation in extracts. Transverse bands were labelled in high-speed supernatants of eggs or oocytes in which replication does not initiate de novo. These patterns corresponded to the patterns of endogenous replication forks in polytene nuclei, monitored by bromodeoxyuridine incorporation in intact salivary glands. By contrast, when nuclei were incubated in low-speed supernatants of eggs, they underwent more extensive chromatin decondensation and initiated replication. The spatial patterns of replication are strikingly different from the endogenous patterns. Instead they closely resemble patterns of clustered replication forks seen in Xenopus sperm nuclei replicating in the extract. This indicates that the egg extract can impose its pattern of replication foci even when the template is presented in the highly organized form of a polytene nucleus.

Animals↗
PubMed1977

A mechanism of duplex DNA replication revealed by enzymatic studies of phage phi X174: catalytic strand separation in advance of replication.

The enzyme system for duplicating the duplex, circular DNA of phage phi X174 (replicative form) in stage II of the replicative life cycle was shown to proceed in two steps: synthesis of the viral (+) strand ]stage II(+)], followed by synthesis of the complementary (-) strand ]stage II(-)] [Eisenberg et al. (1976) Proc. Natl. Acad. Sci. USA 73, 3151-3155]. Novel features of the mechanism of the stage II(+) reaction have now been observed. The product, synthesized in extensive net quantities, is a covalently closed, circular, single-stranded DNA. The supercoiled replicative form I template and three of the four required proteins--the phage-induced cistron A protein (cis A), the host rep protein (rep), and the DNA polymerase III holoenzyme (holoenzyme)--act catalytically; the Escherichia coli DNA unwinding (or binding) protein binds the product stoichiometrically. In a reaction uncoupled from replication, cis A, rep, DNA binding protein, ATP, and Mg2+ separate the supercoiled replicative form I into its component single strands coated with DNA binding protein. In the presence of Mg2+, cis A, nicks the replicative form I; rep, ATP, and Mg2+ achieve strand separation with a concurrent cleavage of ATP and binding of DNA binding protein to the single strands. rep exhibits a single-stranded DNA-dependent ATPase activity. These observations suggest that the rep enzymatically melts the duplex at the replicating fork, using energy provided by ATP; this mechanism may apply to the replication of the E. coli chromosome as well.

Adenosine Triphosphatases↗
PubMed1992

Geminivirus replication proteins are related to prokaryotic plasmid rolling circle DNA replication initiator proteins.

It is demonstrated, by means of computer-assisted analysis, that C1 protein involved in the replication of geminivirus DNA is related to the rolling circle replication initiator proteins of eubacterial plasmids, particularly the plasmids of the pMV158 family. Three sequence motifs conserved in the geminivirus and plasmid replication proteins were delineated, one of them encompassing the Tyr residue that presumably forms a covalent linkage to DNA. These findings are compatible with the results of recent analyses of geminivirus replicative intermediates suggesting a rolling circle mechanism for geminivirus DNA replication. It is hypothesized that C1 protein initiates the rolling circle replication of geminivirus DNA by nicking a specific site in the virus-sense DNA and covalently linking to the 5' side of the nick. The putative rolling circle replication initiator domain comprises the N-terminal portion of C1, whereas its C-terminal part is a putative helicase domain. By analogy with prokaryotic systems, it is speculated that the replication initiator domain and the helicase domain function coordinately. The possibility of the origin of geminiviruses from prokaryotic circular ssDNA replicons is discussed.

Amino Acid Sequence↗
PubMed1979

Plasmid replication functions. III. Origin and direction of replication of a "mini" plasmid derived from R6-5.

Replicating DNA molecules of the mini R6-5 plasmid, pKTO71, were purified by equilibrium centrifugation in two successive ethidium bromide-caesium chloride gradients, converted to linear forms by cleavage with either HindIII or BglII restriction endonuclease, and examined in the electron microscope. Determination of the replication fork positions in 65 replicating molecules demonstrated that replication is initiated at a unique location on the plasmid and that it proceeds uni-directionally from this site. The direction of replication is such that the origin-proximal BglII cleavage site is replicated late or, in the case of the parent R6-5 plasmid, is such that the R-determinant region of the molecule is replicated early. The origin of replication, located by these experiments at R6-5 coordinate 98.6 kb, is clearly distinct from that of the R6-5 incompatibility determinant which has been shown to be located on an adjacent PstI-generated DNA fragment whose termini have R6-5 coordinates 96.8 and 97.9 kb. This result indicates that the incompatibility function is not an origin DNA sequence.

DNA Replication↗
PubMed1975

Initiation of DNA replication in Escherichia coli. II. Effect of rifampicin on the resumption of replication of F episome and chromosome upon the returning of dna mutants from a non-permissive to a permissive temperature.

When E. coli F+ cells carrying the dna-167 or dnaC2 mutation, which causes the temperature-sensitive initiation of DNA replication, are exposed to a non-permissive temperature to stop the replication of chromosome and F factor, and then transferred back to a permissive temperature with the addition of chloramphenicol, one round of the chromosomal replication occurs, but further replication is inhibited. Under these conditions, F DNA replicates coincidentally with the initiation of the chromosomal replication in both strains. When rifampicin is added to the cells upon lowering of the temperature, the chromosome can not replicate in the F+ dna-167 strain, but can do so in the F+ dnaC2 strain. F DNA can replicate in both of the mutant strains under these conditions.

Chloramphenicol↗
PubMed1991

Analysis of replicative intermediates produced during bacteriophage phi 29 DNA replication in vitro.

Replication of bacteriophage phi 29 DNA initiates at either end of its linear double-stranded DNA molecule and proceeds by a strand-displacement mechanism. In the present paper we have used an in vitro phi 29 DNA replication system to analyse by electron microscopy the replicative intermediates produced at different reaction times. Two types of replicative intermediates were observed: type I (full-length double-stranded phi 29 DNA molecules with one or more single-stranded DNA branches) and type II (full-length phi 29 DNA molecules formed by a double-stranded DNA portion of variable length from one end plus a single-stranded DNA portion spanning to the other end). Thus, the types of replicative intermediates produced in vivo were also formed in the in vitro phi 29 DNA replication system. Analysis of type I intermediates indicated that initiation of DNA replication occurs preferentially at both ends of the same DNA template, in a non-simultaneous manner. Type II intermediates appeared as early as two minutes after the reaction started, well before unit-length single-stranded phi 29 DNA molecules were synthesized. In addition, replication of recombinant phi 29 DNA templates lacking terminal protein at one end did not produce type II intermediates and led to an accumulation of full-length single-stranded phi 29 DNA molecules. These two observations strongly suggest that type II intermediates appear when two growing DNA chains, running from opposite ends, merge.

Bacillus subtilis↗
PubMed1979

Inceptor and origin of DNA replication in lambdoid coliphages. I. The lambda DNA minimal replication system.

In a pBR313-lambda dv hybrid plasmid system, stepwise deletion and serial cloning procedures have led to a functional dissection of the DNA replication region of lambdoid bacteriophages lambda, 434 and 21. A simple system for initiation of DNA replication has been detected within lambdoid replicator DNAs, which is active in the absence of several normal replication elements, including the origin of replication (ori) and product of gene O. This "minimal" (or "mini") initiation system depends on the p0 or substitute leftward promoter in conjunction with the newly discovered "inceptor" (ice) element, which is located within the cII gene. Even the fragments containing ori are unable to initiate replication in these hybrid plasmids as long as fragments containing ice are missing. The base sequence of ice resembles transcriptional terminators and it appears to control both termination of primer RNA and inception of daughter strand DNA synthesis. Initiation in the p0-ice mini system of lambda or 21 phages requires the gene P product. Hwever, mini replication of 434 DNA hybrid plasmids required neither O nor P proteins, although there are only two single-base changes in the 434 inceptor sequence. The mini system is repressed by the elements of the maximal lambda replication system, as described in the accompanying publication.

Bacteriophage lambda↗
PubMed1989

Changes in nucleosome repeat lengths precede replication in the early replicating metallothionein II gene region of cells synchronized in early S phase.

Previous investigations showed that inhibition of DNA synthesis by hydroxyurea, aphidicolin, or 5-fluorodeoxyuridine produced large changes in the composition and nucleosome repeat lengths of bulk chromatin. Here we report results of investigations to determine whether the changes in nucleosome repeat lengths might be localized in the initiated replicons, as postulated [D'Anna, J. A., & Prentice, D. A. (1983) Biochemistry 22, 5631-5640]. In most experiments, Chinese hamster (line CHO) cells were synchronized in G1, or they were synchronized in early S phase by allowing G1 cells to enter S phase in medium containing 1 mM hydroxyurea or 5 micrograms mL-1 aphidicolin, a procedure believed to produce an accumulation of initiated replicons that arise from normally early replicating DNA. Measurements of nucleosome repeat lengths of bulk chromatin, the early replicating unexpressed metallothionein II (MTII) gene region, and a later replicating repeated sequence indicate that the changes in repeat lengths occur preferentially in the early replicating MTII gene region as G1 cells enter and become synchronized in early S phase. During that time, the MTII gene region is not replicated nor is there any evidence for induction of MTII messenger RNA. Thus, the results are consistent with the hypothesis that changes in chromatin structure occur preferentially in the early replicating (presumably initiated) replicons at initiation or that changes in chromatin structure can precede replication during inhibition of DNA synthesis. The shortened repeat lengths that precede MTII replication are, potentially, reversible, because they become elongated when the synchronized early S-phase cells are released to resume cell cycle progression.

Animals↗
PubMed1978

The relationship of SV40 replicating chromosomes to two forms of the non-replicating SV40.

SV40 replicating chromosomes were extracted from infected cells using a detergent free extraction method. This procedure also extracts 2 forms of the non-replicating chromosome, one of which corresponds to the well characterized 50-55S SV40 minichromosome. The other is a more compact structure which has a sedimentation coefficient of 80-85S. The replicating chromosomes sediment between the 2 conformations of the mature chromosome. Electron microscopy of the replicating chromosomes suggests an overall conformation that resembles the 50-55S form of the mature chromosome rather than that of the 80-85S structure. Nucleosomes are present on both sides of the replication forks. When the replicating chromosomes were incubated in an in vitro DNA synthesis assay all regions of the SV40 genome were synthesized and a significant fraction of the replicating chromosomes completed replication. The progeny chromosomes co-sedimented with the 50-55S chromosomes which were present prior to the incubation. The sedimentation coefficients and relative amounts of the two forms of the mature chromosome were unaffected by the incubation.

Centrifugation, Density Gradient↗
PubMed1991

Mammalian sex chromosomes. IV. Replication heterogeneity in the late replicating facultative- and constitutive-heterochromatic regions in the X chromosomes of the mole rats, Bandicota bengalensis and Nesokia indica.

The karyotypes of Nesokia indica and Bandicota bengalensis are identical except for their sex chromosomes, which are much larger in Nesokia due to additional constitutive heterochromatin (C.H.). Replication patterns of their sex chromosomes were studied employing 3H-Tdr autoradiography and BrdUrd-FPG staining techniques. Though the "conservative" part of both early- and late-replicating X chromosomes revealed identical replication patterns in most cells, deviant patterns of only the late replicating X chromosome were encountered in approximately 10% cells. Surprisingly, these late-X variants were similar in the two species. The sex chromosome-associated C.H. segments replicated late in S-phase and, in females, the homologous heterochromatin replicated asynchronously--the later replicating one was predominantly associated with the late X. These results suggest structural and functional conservation of the X chromosomes as well as the possible influence of facultative heterochromatin (F.H.) on the replication of associated C.H. in these two species.

Animals↗
PubMed1986

Replication of plasmid RK2 in vitro by a DNA-membrane complex: evidence for initiation of replication and its coupling to transcription and translation.

The following results with an in vitro replication system utilizing a plasmid RK2 DNA-membrane complex indicate that the essential trfA-encoded replication protein of RK2 is present and active in the complex. (i) A complex extracted from a conditional replication mutant of RK2, which contains a temperature-sensitive mutation in trfA, displayed extensive DNA synthesis at the permissive temperature but little activity at the restrictive temperature. A control wild-type RK2 complex showed no inhibition of DNA synthesis at the restrictive temperature. (ii) Analysis of plasmid-encoded proteins revealed that the trfA-specified replication protein and other proteins which may be involved in the replication and maintenance of RK2 are located physically in the complex. Semiconservative plasmid DNA replication by the DNA-membrane complex was indicated by density shift experiments; DNA synthesized in the presence of a heavy-density precursor banded primarily in a heavier-density area of a neutral CsCl density gradient and consisted mostly of heavy- and light-density single-stranded DNA as determined by alkaline CsCl density gradient centrifugation. Plasmid RK2 DNA replication by the DNA-membrane complex appears to be coupled to transcription and translation as indicated by the following results: the inhibitory effects of chloramphenicol on both DNA and protein synthesis by the complex; the stimulation of replication by components normally required for protein synthesis (tRNA and all the common amino acids); the synthesis of RNA and protein by the complex; and the synthesis of specific RK2-encoded proteins.

Bacterial Proteins↗
PubMed1992

Replication of nuclear and mitochondrial DNA in X-ray-damaged cells: evidence for a nuclear-specific mechanism that down-regulates replication.

The mechanism by which X rays inhibit DNA replication has been investigated in three distinct populations of DNA molecules in human cells: (a) large chromosomal DNA, (b) a population of 50-100 10.3-kb nuclear episomal plasmids per cell, and (c) a population of about 500 16-kb cytoplasmic mitochondrial DNA molecules per cell. DNA replication was inhibited by X rays in nuclear chromosomal and plasmid DNA, but not in mitochondrial DNA. The mechanism by which ionizing radiation inhibits DNA replication must therefore be nuclear-specific and is unlikely to involve diffusible low-molecular-weight substances. Since mitochondrial DNA exists in the cell as independent 16-kb circular molecules and responds to radiation as would be expected for small targets, the implication for nuclear plasmids is that their replication is regulated by a large target. A current model for DNA replication involves the movement of DNA through replication centers made up of polymerases, helicases, and associated replication enzymes that are attached to a matrix. The difference in the response to X rays between mitochondrial DNA and nuclear plasmid DNA can be explained if nuclear plasmids are tightly associated with chromosomal DNA and attached to the matrix, and are coordinately replicated.

DNA Replication↗
PubMed1990

Host virus interactions in the initiation of bacteriophage lambda DNA replication. Recruitment of Escherichia coli DnaB helicase by lambda P replication protein.

The bacteriophage lambda P protein promoters replication of the phage chromosome by recruiting a key component of the cellular replication machinery to the viral origin. Specifically, P protein delivers one or more molecules of Escherichia coli DnaB helicase to a nucleoprotein structure formed by the lambda O initiator at the lambda replication origin. Using purified proteins, we have examined the features of the pivotal host virus interaction between P and DnaB. These two proteins interact in vitro to form a P.DnaB protein complex that can be resolved by sedimentation or by chromatography on DEAE-cellulose from the individual free proteins. The sedimentation coefficient of the P.DnaB complex, 13 S, suggests a size larger than that of free DnaB hexamer (Mr = 313,600). The P.DnaB complex isolated by glycerol gradient sedimentation contains approximately three protomers of P/DnaB hexamer, consistent with a molecular weight of 393,000. The isolated P.DnaB complex functions in vitro in the initiation of lambda DNA replication. Interaction of P with DnaB strongly suppressed both the intrinsic DNA-dependent ATPase activity of DnaB, as well as the capacity of DnaB to assist E. coli primase in the general priming reaction. Formation of a P.DnaB protein complex also blocked DnaB from functioning in the initiation of E. coli DNA replication in vitro. The physical and functional properties of lambda P protein suggest that it is a viral analogue of the E. coli DnaC replication protein. Like P, DnaC also binds to DnaB (Wickner, S., and Hurwitz, J. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 921-925), but unlike P, DnaC stimulates DnaB-mediated general priming. When viral P and bacterial DnaC replication proteins were placed in direct competition with one another for binding to DnaB, the viral protein was clearly predominant. For example, a 5-fold molar excess of DnaC protein only partially reversed the inhibitory effect of P on general priming. Furthermore, when a preformed DnaC.DnaB protein complex was incubated briefly with P protein, it was readily converted into a P.DnaB protein complex and the bulk of the bound DnaC was released as free protein. It is likely that the capacity of the lambda P protein to outcompete the analogous host protein for binding to the bacterial DnaB helicase is the critical molecular event enabling infecting phage to recruit cellular replication proteins required for initiation of DNA synthesis at the viral origin.

Bacterial Proteins↗
PubMed1985

Initiation of DNA replication on single-stranded DNA templates catalyzed by purified replication proteins of bacteriophage lambda and Escherichia coli.

Initiation of bacteriophage lambda DNA replication at the chromosomal origin depends on the lambda O and P replication proteins. These two viral initiators, together with an Escherichia coli protein fraction, promote the replication in vitro of single-stranded circular DNA chromosomes such as that of bacteriophage M13. This nonspecific strand initiation reaction, which we have termed the "lambda single-strand replication reaction," has now been established with eight purified proteins, each of which is also required for replication of the phage lambda chromosome in vivo. An early rate-limiting step in the overall reaction is the ATP-dependent assembly of an activated nucleoprotein prepriming complex. In this step the lambda O and P initiators cooperate with the E. coli dnaJ and dnaK proteins to transfer the bacterial dnaB protein onto M13 DNA that is coated with the single-stranded DNA-binding protein. Multiple RNA primers are synthesized on each DNA circle when isolated prepriming complex is incubated with primase and rNTPs. In the complete system, DNA polymerase III holoenzyme extends the first primer synthesized into full-length complementary strands. Because the properties of this system are closely analogous to those found for the replication of phi X174 viral DNA by E. coli proteins, we infer that a mobile prepriming or priming complex (primosome) operates in the lambda single-strand replication reaction.

Bacterial Proteins↗
PubMed1989

Purification of replication protein C, a cellular protein involved in the initial stages of simian virus 40 DNA replication in vitro.

The replication of simian virus 40 (SV40) DNA is dependent upon a single viral protein [tumor (T) antigen] and multiple cellular proteins. To define the required cellular proteins, we have made use of a cell-free system that supports the replication of plasmid DNA molecules containing the SV40 origin of replication. We report here the purification from HeLa cell extracts of replication protein C (RP-C), a previously undescribed protein that is required to reconstitute efficient DNA replication in vitro. Highly purified preparations of RP-C contain two closely related polypeptides of 32 and 34 kDa. Preincubation of purified RP-C with T antigen and the DNA template largely eliminates the delay normally observed before the onset of rapid DNA synthesis. In addition, RP-C stimulates the unwinding of duplex DNA molecules containing the SV40 replication origin in a reaction that requires T antigen and a single-stranded DNA binding protein. These observations suggest that RP-C is involved in the initial steps of SV40 DNA replication in vitro.

Antigens, Polyomavirus Transforming↗
PubMed1992

Identification of nuclear pre-replication centers poised for DNA synthesis in Xenopus egg extracts: immunolocalization study of replication protein A.

We demonstrate by immunofluorescence that a 70-kD protein (P70) purified from Xenopus egg extracts is associated with subnuclear foci (about 200) which we propose to be an assembly of DNA pre-replication centers (preRCs). A cDNA encoding this protein reveals that P70 is the Xenopus homologue of replication protein A (RPA also called RF-A). RPA is know to be a cellular, three-subunit single-stranded DNA binding protein, which assists T-antigen in the assembly of the pre-priming complex in the SV40 replication system. The punctated preRCs exist transiently; they form post-mitotically during the period of nuclear membrane breakdown and disappear during ongoing DNA replication. P70 is homogeneously associated with chromatin at the later stages of the S-phase and is displaced from chromatin post replication, so that P70 cannot be detected on mitotic chromosomes. Double-immunofluorescence studies using biotin-dUTP demonstrate that initiation of DNA synthesis is confined to preRCs, resulting in the punctated replication pattern observed previously by others. PreRCs form efficiently on decondensed chromatin in membrane-free egg extracts if ATP and divalent cations are present. Our results suggest that preRCs are composed of an assembly of a large number of pre-initiation replication complexes poised for initiation at discreet subnuclear regions prior to nuclear reconstruction and initiation of DNA synthesis.

Amino Acid Sequence↗
PubMed1987

5-aza-C-induced changes in the time of replication of the X chromosomes of Microtus agrestis are followed by non-random reversion to a late pattern of replication.

Treatment with 5-azacytidine (5-aza-C) causes an advance in the time of replication and enhances the DNase-I sensitivity of the inactive X chromosome in Gerbillus gerbilllus fibroblasts. We found that these changes were not stably inherited and upon removal of the drug the cells reverted to the original state of one active and one inactive X chromosome. In order to determine whether this reversion was random, we used a cell line of female Microtus agrestis fibroblasts in which the two X chromosomes are morphologically distinguishable. In this work we show that the reversion to a late pattern of replication is not random, and the originally late replicating X chromosome is preferentially "reinactivated", suggesting an imprinting-like marking of one or both X chromosomes. The changes in the replication pattern of the X chromosome were associated with changes in total DNA methylation. Double treatment of cells with 5-aza-C did not alter this pattern of euchromatin activation and reinactivation. A dramatic advance in the time of replication of the entire X linked constitutive heterochromatin (XCH) region was however, observed in the doubly treated cells. This change in the replication timing of the XCH occurred in both X chromosomes and was independent of the changes observed in the euchromatic region. These observations suggest the existence of at least two independent regulatory sites which control the timing of replication of two large chromosomal regions.

Animals↗