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PubMed1985

Replication control in promiscuous plasmid RK2: kil and kor functions affect expression of the essential replication gene trfA.

We previously reported that broad-host-range plasmid RK2 encodes multiple host-lethal kil determinants (kilA, kilB1, kilB2, and kilC) which are controlled by RK2-specified kor functions (korA, korB, and korC). Here we show that kil and kor determinants have significant effects on RK2 replication control. First, korA and korB inhibit the replication of certain RK2 derivatives, unless plasmid replication is made independent of the essential RK2 gene trfA. Second, kilB1 exerts a strong effect on this interaction. If the target plasmid is defective in kilB1, sensitivity to korA and korB is enhanced at least 100-fold. Thus, korA and korB act negatively on RK2 replication, whereas kilB1 acts in a positive manner to counteract this effect. A mutant RK2 derivative, resistant to korA and korB, was found to have fused a new promoter to trfA, indicating that the targets for korA and korB are at the 5' end of the trfA gene. We constructed a trfA-lacZ fusion and found that synthesis of beta-galactosidase is inhibited by korA and korB. Thus korA, korB, and kilB1 influence RK2 replication by regulating trfA expression. We conclude that the network of kil and kor determinants is part of a replication control system for RK2.

Bacterial Proteins↗

PubMed1971

Deoxyribonucleic acid replication in simian virus 40-infected cells. IV. Two different requirements for protein synthesis during simian virus 40 deoxyribonucleic acid replication.

The replication of simian virus 40 (SV40) deoxyribonucleic acid (DNA) was inhibited by 99% 2 hr after the addition of cycloheximide to SV40-infected primary African green monkey kidney cells. The levels of 25S (replicating) and 21S (mature) SV40 DNA synthesized after cycloheximide treatment were always lower than those observed in an infected untreated control culture. This is consistent with a requirement for a protein(s) or for protein synthesis at the initiation step in SV40 DNA replication. The relative proportion of 25S DNA as compared with 21S viral DNA increased with increasing time after cycloheximide treatment. Removal of cycloheximide from inhibited cultures allowed the recovery of viral DNA synthesis to normal levels within 3 hr. During the recovery period, the ratio of 25S DNA to 21S DNA was 10 times higher than that observed after a 30-min pulse with (3)H-thymidine with an infected untreated control culture. The accumulation of 25S replicating SV40 DNA during cycloheximide inhibition or shortly after its removal is interpreted to mean that a protein(s) or protein synthesis is required to convert the 25S replicating DNA to 21S mature viral DNA. Further evidence of a requirement for protein synthesis in the 25S to 21S conversion was obtained by comparing the rate of this conversion in growing and resting cells. The conversion of 25S DNA to 21S DNA took place at a faster rate in infected growing cells than in infected confluent monolayer cultures. A temperature-sensitive SV40 coat protein mutation (large-plaque SV40) had no effect on the replication of SV40 DNA at the nonpermissive temperature.

Cell Line↗

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↗

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↗

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↗

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↗

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↗

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↗

PubMed1992

Topoisomerase I is preferentially associated with normal SV40 replicative intermediates, but is associated with both replicating and nonreplicating SV40 DNAs which are deficient in histones.

Based on the use of equilibrium centrifugation in CsCl to separate covalent complexes between topoisomerase I and DNA from protein-free DNA, it was concluded previously that the topoisomerase is preferentially associated with replicating SV40 DNA (Champoux, J. J. 1988. J. Virol. 62:3675-3683). One explanation for the failure to find the enzyme associated with nonreplicating viral DNA is that most of the completed DNA is rapidly sequestered for encapsidation and inaccessible to topoisomerase I. This explanation has been ruled out in the present work by the finding that topoisomerase I in COS-1 cells is also preferentially associated with the replicative form of an SV40 origin-containing plasmid that lacks the genes coding for the virion structural proteins and therefore cannot be encapsidated. Thus it appears that some structural feature of the replicating DNA or the replication complex specifically recruits the topoisomerase to the DNA. SV40 DNA which is produced in the presence of the protein synthesis inhibitor, puromycin, is deficient in histones and as a result lacks normal chromatin structure. Topoisomerase I was found to be associated with SV40 DNA under these conditions whether or not it was replicating. This observation is interpreted as an indication that under normal conditions, chromatin structure limits access of topoisomerase I to the nonreplicating viral DNA.

Animals↗

PubMed1978

Okazaki pieces grow opposite to the replication fork direction during simian virus 40 DNA replication.

Simian virus 40 replicating DNA was pulse labeled with alpha-32P-dATP using an acellular DNA replication system. Nascent DNA chains of less than 200 nucleotides (Okazaki pieces) were then isolated from the denatured replicating DNA by electrosieving through a polyacrylamide gel column. The purified Okazaki pieces were hybridized to separated strands of Bg1(1)+Hpa1 simian virus 40 DNA restriction fragments immobilized on nitrocellulose filters. Only strands with polarity of the DNA replication fork direction hybridized with Okazaki pieces. Hence, Okazaki pieces in simian virus 40 are synthesized against the DNA replication fork direction.

DNA Replication↗

PubMed1973

Studies on the mechanism of replication of adenovirus DNA. II. The nature of single-stranded DNA in replicative intermediates.

Replicative intermediates of adenovirus type 5 DNA contain large stretches of single-stranded DNA. We have shown that this single-stranded DNA is mainly of parental origin, whereas all new DNA synthesized during one round of replication has a double-stranded structure. Hybridization experiments of the single-stranded DNA with isolated complementary strands of adenovirus type 5 DNA showed that this DNA hybridized only with the viral L-strand (the strand with the lower equilibrium density in alkaline CsCl) indicating that it represents the viral H-strand. This observation implies that replication always starts from one and the same molecular end. Electron microscopy of partially denatured Y-shaped intermediates confirmed this and showed that replication started from the molecular right end (the end richest in A-T base pairs). In conclusion, we have shown that replication of adenovirus type 5 DNA starts at the molecular right end, displacing the parental H-strand.

Adenosine Triphosphate↗

PubMed1974

Role of genetic recombination in DNA replication of bacteriophage lambda. II. Effect in DNA replication by gene delta.

We have studied the effect of delta mutations in phage lambda on DNA synthesis as assayed by the accumulation of lambda DNA in infected cells. We find that delta mutants appear to generate somewhat less DNA than lambda(+) in a rec(+) host, suggesting the wild-type delta gene may act in DNA replication. An additional clue to delta function arises if replication is measured in the gamma-negative situation where concatemer formation is abortive. In this situation, the wild-type delta gene has an "inhibitory" effect on replication. A similar inhibitory effect on replication due to delta is observed after infection of P(2) lysogens. We conclude from these studies that the delta gene may act with alpha, beta, and gamma genes, possibly in a process affecting DNA replication.

Coliphages↗

PubMed1977

Replication process of the parvovirus H-1. VII. Electron microscopy of replicative-form DNA synthesis.

The geometry of replicative form (RF) DNA synthesis of the H-1 parvovirus was studied with the electron microscope using formamide or aqueous variations of the Kleinschmidt spreading procedure. H-1 DNA was isolated from human or hamster cells infected with a temperature-sensitive mutant, ts1, which is deficient in progeny single-stranded DNA synthesis at the restrictive temperature (S.L. Rhode, 1976), thus minimizing possible confusion between RF and progeny DNA replicative intermediates (RIs). The purity of the isolated H-1 DNA, as determined by gel electrophoresis, ethidium bromide staining, autoadiography, and digestion with endo R-EcoRI, was high. H-1 RF DNA'S WERE LINEAR DOUBLE-STRANDED MOLECULES, 1.53 MUM IN LENGTH. H-1 RIs of RF DNA replication were double-stranded, Y-shaped molecules, with the same length as RF DNAs. The replication origin was localized no more than 0.15 genome lengths from one end of the RF DNA, with replication proceeding toward the other end at a uniform rate. Similar RF and RI molecules of dimer size were also observed. The length of H-1 single-stranded DNA extracted from purified virions was measured relative to that of phiX174 and it had a very similar contour length, so that the molecular weight of H-1 single-stranded DNA would be at least 1.48 X 10(6) to 1.59 X 10(6) (Berkowitz and Day, 1974).

Cell Line↗