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At least 271 records · Page 15Linked to original sources
PubMed1986

Initiation of simian virus 40 DNA replication in vitro: aphidicolin causes accumulation of early-replicating intermediates and allows determination of the initial direction of DNA synthesis.

Aphidicolin, a specific inhibitor of DNA polymerase alpha, provided a novel method for distinguishing between initiation of DNA synthesis at the simian virus 40 (SV40) origin of replication (ori) and continuation of replication beyond ori. In the presence of sufficient aphidicolin to inhibit total DNA synthesis by 50%, initiation of DNA replication in SV40 chromosomes or ori-containing plasmids continued in vitro, whereas DNA synthesis in the bulk of SV40 replicative intermediate DNA (RI) that had initiated replication in vivo was rapidly inhibited. This resulted in accumulation of early RI in which most nascent DNA was localized within a 600- to 700-base-pair region centered at ori. Accumulation of early RI was observed only under conditions that permitted initiation of SV40 ori-dependent, T-antigen-dependent DNA replication and only when aphidicolin was added to the in vitro system. Increasing aphidicolin concentrations revealed that DNA synthesis in the ori region was not completely resistant to aphidicolin but simply less sensitive than DNA synthesis at forks that were farther away. Since DNA synthesized in the presence of aphidicolin was concentrated in the 300 base pairs on the early gene side of ori, we conclude that the initial direction of DNA synthesis was the same as that of early mRNA synthesis, consistent with the model proposed by Hay and DePamphilis (Cell 28:767-779, 1982). The data were also consistent with initiation of the first DNA chains in ori by CV-1 cell DNA primase-DNA polymerase alpha. Synthesis of pppA/G(pN)6-8(pdN)21-23 chains on a single-stranded DNA template by a purified preparation of this enzyme was completely resistant to aphidicolin, and further incorporation of deoxynucleotide monophosphates was inhibited. Therefore, in the presence of aphidicolin, this enzyme could initiate RNA-primed DNA synthesis at ori first in the early gene direction and then in the late gene direction, but could not continue DNA synthesis for an extended distance.

Animals↗
PubMed1999

Tumor suppressor protein WT1 inhibits autonomous DNA replication directly, as well as indirectly by causing loss of replicated DNA as a consequence of cell death induced by the protein.

The effects of the Wilms' tumor suppressor protein WT1 on autonomous DNA replication under stable transfection conditions were investigated. COS ts2 monkey kidney cells, which express the simian virus 40 (SV40) replication initiator protein large tumor antigen (TAg) as a temperature-sensitive protein, were stably transfected with SV40 origin-containing plasmids expressing WT1 from the Zn2+ and Cd2+-inducible metallothionein promoter. Stable transformant clones of cells containing the integrated plasmids were isolated at the non-permissive temperature, expanded, and shifted to the permissive temperature to allow autonomous replication of the plasmid and overexpression of WT1. Expression of WT1 triggered apoptosis of the cells. Analysis of the kinetics of occurrence of cell death and accumulation of the replicated plasmid indicated that WT1 inhibited replication directly, and also indirectly by causing loss of replicated plasmid as a consequence of WT1-induced cell death.

Animals↗
PubMed1992

Initiation of DNA replication in ColE1 plasmids containing multiple potential origins of replication.

We have investigated the frequency of replication origin usage in bacterial plasmids containing more than one potential origin. Escherichia coli recA- cells were selectively transformed with pBR322 monomers, dimers, or trimers. Plasmid DNA was isolated and digested with a restriction enzyme that cut the monomer only once, and the replicative intermediates (RIs) were analyzed by neutral/neutral two-dimensional agarose gel electrophoresis. Evidence for initiation outside the linearized plasmid was found only for oligomers. Moreover, in dimers, the intensity of the signal indicative for external initiation was equivalent to that reflecting internal initiation, whereas it was approximately twice as strong in trimers. To determine whether initiation could occur simultaneously at two origins in a single plasmid, we studied the replication of a neodimer in which both units could be unambiguously distinguished. The results showed that although both origins were equally competent to initiate replication, only one was active per plasmid. These observations strongly suggest that in ColE1 plasmids, replication initiates at a single site even when there are several identical potential origins per plasmid. In addition to the conventional two-dimensional gel patterns, novel specific patterns were observed with intensities that varied from one DNA sample to another. These unique patterns were the result of breakage of the RIs at a replication fork. This type of breakage changes both the mass and shape of RIs. When the entire population of RIs is affected, a new population of molecules is formed that may generate a novel pattern in two-dimensional gels.

DNA Replication↗
PubMed1989

Ordered assembly of nucleoprotein structures at the bacteriophage lambda replication origin during the initiation of DNA replication.

Replication of the chromosome of bacteriophage lambda depends on the cooperative action of two phage-coded proteins and seven replication and heat shock proteins from its Escherichia coli host. As previously described, the first stage in this process is the binding of multiple copies of the lambda O initiator to the lambda replication origin (ori lambda) to form the nucleosomelike O-some. The O-some serves to localize subsequent protein-protein and protein-DNA interactions involved in the initiation of lambda DNA replication to ori lambda. To study these interactions, we have developed a sensitive immunoblotting protocol that permits the protein constituents of complex nucleoprotein structures to be identified. Using this approach, we have defined a series of sequential protein assembly and protein disassembly events that occur at ori lambda during the initiation of lambda DNA replication. A second-stage ori lambda.O (lambda O protein).P (lambda P protein).DnaB nucleoprotein structure is formed when O, P, and E. coli DnaB helicase are incubated with ori lambda DNA. In a third-stage reaction the E. coli DnaJ heat shock protein specifically binds to the second-stage structure to form an ori lambda.O.P.DnaB.DnaJ complex. Each of the nucleoprotein structures formed in the first three stages was isolated and shown to be a physiological intermediate in the initiation of lambda DNA replication. The E. coli DnaK heat shock protein can bind to any of these early stage nucleoprotein structures, and in a fourth-stage reaction a complete ori lambda.O.P.DnaB.DnaJ.DnaK initiation complex is assembled. Addition of ATP to the reaction enables the DnaK and DnaJ heat shock proteins to mediate a partial disassembly of the fourth-stage complex. These protein disassembly reactions activate the intrinsic helicase activity of DnaB and result in localized unwinding of the ori lambda template. The protein disassembly reactions are described in the accompanying articles.

Bacteriophage lambda↗
PubMed1986

Replication of kinetoplast DNA in isolated kinetoplasts from Crithidia fasciculata. Identification of minicircle DNA replication intermediates.

The kinetoplast DNA (kDNA) of trypanosomes is comprised of thousands of DNA minicircles and 20-50 maxicircles catenated into a single network. We show that kinetoplasts isolated from the trypanosomatid species Crithidia fasciculata incorporate labeled nucleotides and support minicircle DNA replication in a manner which mimics two characteristics of minicircle replication in vivo: 1) the minicircles are replicated as free molecules and subsequently reattached to the kDNA network, and 2) a replication intermediate having a structure consistent with a highly gapped minicircle species is generated. In addition, a class of minicircle DNA replication intermediates is observed containing discontinuities at specific sites within each of the newly synthesized DNA strands. By using a strain of C. fasciculata possessing nearly homogenous minicircles, we were able to map the discontinuities to two small regions situated 180 degrees apart on the minicircle. Each region has two sites at which a discontinuity can occur, one on each strand and separated by approximately 100 base pairs. These sites may represent origins of minicircle DNA replication.

Animals↗
PubMed1984

Protein-primed replication of plasmids containing the terminus of the adenovirus genome. II. Purification and characterization of a host protein required for the replication of DNA templates devoid of the terminal protein.

A host protein, which is required for the replication of a plasmid DNA (pLA1), has been purified from extracts of uninfected HeLa nuclei. This plasmid DNA contains the origin of adenovirus DNA replication but lacks the 55,000-dalton terminal proteins. The purified host protein has been designated factor pL. Factor pL is essential for the initiation of DNA replication of EcoRI-digested pLA1 DNA, which proceeds via the formation of a covalent complex between the 80,000-dalton adenovirus coded preterminal protein and 5' dCMP. Factor pL has been purified approximately 120-fold to greater than 75% homogeneity. It is a heat labile and N-ethylmaleimide-sensitive protein with a native Mr = 39,000 (+/- 2,000). Initiation of DNA replication using EcoRI-digested pLA1 DNA as the template requires the 80,000-dalton preterminal protein and the 140,000-dalton adenovirus DNA polymerase, in addition to factor pL, and is stimulated as much as 10-fold by nuclear factor I ( Nagata , K., Guggenheimer , R. A., Enomoto , T., Lichy , J. H., and Hurwitz , J. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 6438-6442). Factor pL has no effect on in vitro DNA replication when adenovirus DNA covalently linked to the 55,000-dalton terminal protein is used as the template, however the replication of adenovirus DNA treated with Pronase, becomes totally dependent upon the addition of factor pL.

Adenoviridae↗
PubMed1981

Evidence for double replication of chromosomal DNA segments as a general consequence of DNA replication inhibition.

We have previously presented evidence that a transient inhibition of DNA synthesis by a pulse of 1-beta-D-arabinofuranosylcytosine (ara-C) results in a disruption of the pattern of replication of the chromosomal DNA of cultured human cells, resulting in some DNA segments being replicated more than once in a single S phase. Further evidence is presented in this paper that this effect is not a specific property of the ara-C molecule in that a similar effect is produced in cells by a pulse of 9-beta-D-arabinofuranosyladenine (ara-A) and also by a pulse of cycloheximide. The activated form of ara-A and ara-C (the triphosphates) both inhibit DNA synthesis at the level of the polymerase. Double replication following an ara-A pulse demonstrates that double replication after an ara-C pulse is not caused by some specific property of the ara-C molecule which might be unrelated to any effect on DNA synthesis. However, cycloheximide is an inhibitor of mammalian protein synthesis and inhibits DNA synthesis only indirectly, probably through a consequent deficiency of DNA-packaging proteins. Hence, the occurrence of double replication of chromosomal DNA segments following a pulse of cycloheximide is consistent with this phenomenon being a general and nonspecific consequence of the freezing of DNA replication forks.

Cell Line↗
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↗
PubMed1981

Plasmid replication functions. VII. Electron microscopic localization of RNA polymerase binding sites in the replication control region of plasmid R6-5.

RNA polymerase binding sites on the R6-5 miniplasmid derivative, plasmid pKT401, were mapped by electron microscopy of DNA:RNA polymerase complexes formed with both circular-supercoiled and restriction endonuclease-linearized plasmid DNA molecules. Of eight specific binding sites on pKT401 that were identified, three were found to be in the P-6 fragment of the plasmid replication region, three in the Tn3 element, and two in other parts of the plasmid molecule. Binding sites 1 and 3 in the P-6 fragment are most probably the promoters of the copB and copA/incA plasmid replication control genes, respectively, whereas site 2 in this fragment appears to be the promoter of the essential replication gene, repA. The location of these promoters in relation to the site of action of the plasmid replication control elements, copT, and the origin of replication, oriV, suggests that replication control may be effected by regulation of transcription events initiated at site 2, or of the activity of transcripts initiated from this site, i.e., by regulation of the expression of the repA gene or another function dependent upon these events.

Binding Sites↗
PubMed1995

Functional cDNA clones of the human respiratory syncytial (RS) virus N, P, and L proteins support replication of RS virus genomic RNA analogs and define minimal trans-acting requirements for RNA replication.

The RNA-dependent RNA polymerase of human respiratory syncytial (RS) virus was expressed in a functional form from a cDNA clone. Coexpression of the viral polymerase (L) protein, phosphoprotein (P), and nucleocapsid (N) protein allowed us to develop a system for expression and recovery of replicable RS virus RNA entirely from cDNA clones. cDNA clones of the N, P, and L genes were constructed in pGEM-based expression plasmids and shown to direct expression of the appropriate polypeptides. Two types of RS virus genomic RNA analogs were expressed from an intracellular transcription plasmid that directed the synthesis of RNAs with defined 5' and 3' ends. One analog included the authentic 5' and 3' termini of the genome, and the second contained the authentic 5' terminus and its complement at the 3' terminus as found in copyback defective interfering RNAs of other negative-strand RNA viruses. Both types of genomic analogs were encapsidated and replicated in cells expressing the RS virus N, P, and L proteins. Omission of any of the three viral proteins abrogated replication, thereby defining the N, P, and L proteins as the minimal trans-acting proteins required for RNA replication. This system has the advantages that expression occurs at a level sufficient to allow direct biochemical analysis of the products of RNA replication and that neither the use of reporter genes nor wild-type RS helper virus is required. These features allow analysis of both cis- and trans-acting factors involved in the control of replication of RS virus RNA.

Cell Line↗
PubMed1992

cdc2 family kinases phosphorylate a human cell DNA replication factor, RPA, and activate DNA replication.

RPA is a single-stranded DNA binding protein complex purified from human cells and is essential for the initiation and elongation stages of SV40 DNA replication in vitro. In both human and yeast cells, the 34 kDa polypeptide subunit of RPA is phosphorylated in the S and G2 phases of the cell cycle and not in G1. One of the major RPA kinases present in extracts of human cells was purified and shown to be the cyclin B-cdc2 complex. This purified kinase, and a closely related cyclin A associated cdc2-like kinase, phosphorylated RPA p34 on a subset of the chymotryptic peptides that were phosphorylated in vivo at the G1-S transition. Two serines near the N-terminus of RPA p34 were identified as possible sites of phosphorylation by cdc2 kinase. These same serines were necessary for RPA phosphorylation in vivo. The purified cdc2 kinase stimulated SV40 DNA replication in vitro when added to G1 cell extracts. The kinase also stimulated unwinding at the origin of replication, one of the earliest steps in DNA replication requiring RPA, but only in the presence of an additional factor present in G1 cell extracts. Thus, one or more members of the cyclin-cdc2 kinase family may be required for the initiation and maintenance of S phase, in part due to their ability to phosphorylate and activate a cellular DNA replication factor, RPA.

Adenovirus Early Proteins↗
PubMed1994

Replication intermediates of rice tungro bacilliform virus DNA support a replication mechanism involving reverse transcription.

Rice tungro bacilliform virus (RTBV) replication intermediates have been studied in rice plants infected with the virus. Unencapsidated virus-specific molecules were identified which had open circular, linear, supercoiled (SC), strong-stop, single-stranded, linear double-stranded hairpin, and double-stranded with single-stranded extension DNA forms. The structures of these different DNA forms were consistent with the replication model of cauliflower mosaic virus and support other results that reverse transcription is involved in the replication of RTBV. The existence of nonspecific and defective (+)-strand priming is suggested. The relative amount of SC DNAs differs in various tissues of the same plant and in the same tissue at different ages. This indicates host regulation of the virus replication cycle and a feedback regulatory mechanism in controlling the SC DNA level. There are no obvious differences in the composition of the replication intermediates between insect-infected and agroinoculated rice plants.

DNA Replication↗
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↗
PubMed1989

Evidence for multiple vegetative DNA replication origins and alternative replication mechanisms of bovine papillomavirus type 1.

By following up the chance detection in the electron microscope of a DNA replication intermediate within a preparation of bovine papillomavirus (BPV-1) DNA isolated from purified virus particles, information was obtained about the mechanism of BPV-1 genome replication during the final stages of virus multiplication in naturally infected bovine wart tissue. The structure of viral replication intermediates was investigated by electron microscopic analysis of viral DNA linearized by digestion with restriction endonucleases which cleave the circular BPV-1 chromosome at defined sites. Both Cairns and rolling circle-type molecules were identified. Furthermore, replication eyes were widely distributed within the viral genome, indicating that vegetative BPV-1 DNA replication origins are largely uncoupled from previously described plasmid maintenance sequence elements.

Bovine papillomavirus 1↗
PubMed1990

DNA replication studies with coliphage 186. III. A single phage gene is required for phage 186 replication.

We have shown that the BglII to BamHI (79.6% to 95.8%) region of the coliphage 186 chromosome can direct 186-specific replication. DNA sequencing of the region revealed five presumptive genes, CP80, CP81, CP83, CP84 and CP87. Surprisingly, alleles of the previously defined replication gene, A, were localized in both CP84 and CP87. We have successfully constructed a 186 minichromosome using the single gene CP87, and determined that CP84 was not concerned with replication, neither of a minichromosome nor of the phage. Rather, the replication defect seen with amber mutants of CP84 reflects a polarity effect on the downstream expression of CP87. We have concluded that CP87 is the only phage gene necessary for 186 replication, and have called it gene A.

Amino Acid Sequence↗
PubMed2001

Regulation of herpes simplex virus type 1 replication in Vero cells by Psychotria serpens: relationship to gene expression, DNA replication, and protein synthesis.

Inhibitory effects of ethanolic extracts from seven Chinese herbs on herpes simplex virus type 1 (HSV-1) replication were investigated. From a bioassay-guided fractionation procedure, PS-A-6 was isolated from Psychotria serpens (P. serpens), which suppressed HSV-1 multiplication in Vero cells without apparent cytotoxicity. Time-of-addition experiments suggested that the inhibitory action of PS-A-6 on HSV-1 replication was not through blocking of virus adsorption. In an attempt to further localize the point in the HSV-1 replication cycle where arrest occurred, a set of key regulatory events leading to viral multiplication was examined, including viral gene expression, DNA replication, and structural protein synthesis. The results indicated that gB mRNA and protein expression in Vero cells were impeded by PS-A-6. Southern blot analysis showed that HSV-1 DNA replication in Vero cells was arrested by PS-A-6. In addition, PS-A-6 decreased thymidine kinase (tk) and ICP27 mRNA expression in the cells. The mechanisms of antiviral action of PS-A-6 seem to be mediated, at least in part, through inhibition of early transcripts of HSV-1, such as tk and ICP27 mRNAs, arresting HSV-1 DNA synthesis and gB gene expression in Vero cells. Plans are underway for the isolation of pure compounds from PS-A-6 and elucidation of their mechanism of action.

Animals↗
PubMed2001

Expression analysis using DNA microarrays demonstrates that E2F-1 up-regulates expression of DNA replication genes including replication protein A2.

The transcription factor E2F-1 plays a pivotal role in the regulation of G1/S transition in higher eukaryotes cell cycle. We used a cell line containing an inducible E2F-1 and oligonucleotide microarray analysis to identify novel E2F target genes. We show that E2F-1 up-regulates the expression of a number of genes coding for components of the DNA replication machinery. Among them is the gene coding for the 32 Kd subunit of replication protein A (RPA2). Replication protein A is the most abundant single strand DNA binding complex and it is essential for DNA replication. We demonstrate that RPA2 is a novel E2F target gene whose expression can be directly regulated by E2F-1 via E2F binding sites in its promoter. In addition, expression of Topoisomerase IIalpha and subunit IV of DNA polymerase alpha is also up-regulated upon E2F-1 induction. Taken together, these results provide novel links between components of the DNA replication machinery and the cell growth regulatory pathway involving the Rb tumor suppressor and E2F.

Antigens, Neoplasm↗
PubMed1991

Replicational release of geminivirus genomes from tandemly repeated copies: evidence for rolling-circle replication of a plant viral DNA.

Agrobacterium-mediated inoculation of Nicotiana benthamiana plants with Ti plasmids containing tandem genome repeats derived from different strains of the gemini-virus beet curly top virus (BCTV) resulted in the production of unit-length recombinant progeny genomes in systemically infected plants. When two putative plus-strand origins of replication were present in constructs used as inocula, a replicational escape mechanism was favored that resulted in progeny genomes of a single predominant genotype. The genotype was dependent upon the arrangement of repeated parental genomes in the inocula. Sequencing across the junction between parental BCTV strains in the recombinant progeny allowed mapping of the plus-strand origin of replication to a 20-base-pair sequence within the conserved hairpin found in all geminivirus genomes. In contrast, when inocula contained tandemly repeated BCTV genome sequences but only a single conserved hairpin, a number of different progeny genotypes were simultaneously replicated in infected plants, a result expected if unit-length viral genomes were generated by random intramolecular recombination events. These results and other considerations indicate that geminivirus DNA replication occurs by a rolling-circle mechanism.

Base Sequence↗