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PubMed1987

Mechanism of replication of bacteriophage phi X174. XXII. Site-specific mutagenesis of the A* gene reveals that A* protein is not essential for phi X174 DNA replication.

The A and A* proteins of phage phi X174 are encoded in the same reading frame in the viral genome; the smaller A protein is the result of a translational start signal with the A gene. To differentiate their respective functions, oligonucleotide-directed site-specific mutagenesis was used to change the ATG start codon of the phi X 174 A* gene, previously cloned into pCQV2 under lambda repressor control, into a TAG stop codon. The altered A gene was then inserted back into phi X replicative form DNA to produce an amber mutant, phi XamA*. Two different Escherichia coli amber suppressor strains infected with this mutant produced viable progeny phage with only a slight reduction in yield. In Su+ cells infected with phi XamA*, phi X gene A protein, altered at one amino acid, was synthesized at normal levels; A* protein was not detectable. These observations indicate that the A* protein increases the replicative efficiency of the phage, perhaps by shutting down host DNA replication, but is not required for replication of phi X174 DNA or the packaging of the viral strand under the conditions tested.

Bacteriophage phi X 174↗

PubMed1987

Simian virus 40 large tumor antigen requires three core replication origin domains for DNA unwinding and replication in vitro.

Simian virus 40 (SV40) large tumor antigen (T antigen) unwinds DNA containing the SV40 origin of replication. The origin requirement for unwinding can be satisfied by the 64-base-pair SV40 core origin that supports T-antigen-dependent DNA replication both in vivo and in vitro. The core origin contains three domains with specific DNA sequence features. These include an inverted repeat, a central T-antigen binding domain, and an adenine- and thymine-rich domain containing a DNA bending focus. The domain and spacer requirements of the core origin for DNA unwinding and replication in vitro are strikingly similar to the origin requirements for DNA replication in vivo. Thus, each of the three functional domains of the core origin contributes directly to the initiation of duplex DNA unwinding by T antigen.

DNA Helicases↗

PubMed1975

Replication of bacteriophage M13 IX. Requirement of the Escherichia coli dnaG function for M13 duplex DNA replication.

Temperature-shift experiments with an Escherichia coli dnaG strain indicate a requirement for the dnaG function for M13 phage production only at an early stage of infection. Mutant cells infected at nonpermissive temperature form the parental RF (SS leads to RF) but do not replicate further. A shift to nonpermissive temperature after infection inhibits RF leads to RF replication but not RF leads to SS synthesis. The synthesis of both strands of the duplex RF was inhibited equally after a temperature shift during RF leads to RF replication. We infer that the dnaG protein is required for M13 production only during RF replication and that it is required for the synthesis of both strands of the RF.

Centrifugation, Density Gradient↗

PubMed1991

Replication factors required for SV40 DNA replication in vitro. II. Switching of DNA polymerase alpha and delta during initiation of leading and lagging strand synthesis.

Replication factors A and C (RF-A and RF-C) and the proliferating cell nuclear antigen (PCNA) differentially augment the activities of DNA polymerases alpha and delta. The mechanism of stimulation by these replication factors was investigated using a limiting concentration of primed, single-stranded template DNA. RF-A stimulated polymerase alpha activity in a concentration-dependent manner, but also suppressed nonspecific initiation of DNA synthesis by both polymerases alpha and delta. The primer recognition complex, RF-C.PCNA.ATP, stimulated pol delta activity in cooperation with RF-A, but also functioned to prevent abnormal initiation of DNA synthesis by polymerase alpha. Reconstitution of DNA replication with purified factors and a plasmid containing the SV40 origin sequences directly demonstrated DNA polymerase alpha dependent synthesis of lagging strands and DNA polymerase delta/PCNA/RF-C dependent synthesis of leading strands. RF-A and the primer recognition complex both affected the relative levels of leading and lagging strands. These results, in addition to results in an accompanying paper (Tsurimoto, T., and Stillman, B. (1991) J. Biol. Chem. 266, 1950-1960), suggest that an exchange of DNA polymerase complexes occurs during initiation of bidirectional DNA replication at the SV40 origin.

Adenosine Triphosphate↗

PubMed1988

Initiation of adenovirus DNA replication. I. Mechanism of action of a host protein required for replication of adenovirus DNA templates devoid of the terminal protein.

The in vitro replication of adenovirus (Ad) DNA covalently attached to the 55-kDa terminal protein requires at least five proteins including the 80-kDa preterminal protein, the Ad DNA polymerase, the Ad DNA binding protein, nuclear factor I, and topoisomerase I. The replication of Ad DNA templates devoid of the terminal protein requires an additional protein, designated factor pL, which has been purified from uninfected HeLa cell nuclei (Guggenheimer, R. A., Nagata, K., Kenny, M., and Hurwitz, J. (1984) J. Biol. Chem. 259, 7815-7825). Factor pL has been found to contain an intrinsic 5'----3' exonuclease activity. When Ad DNA templates lacking the terminal protein were pretreated with factor pL, the requirement for factor pL in the replication reaction was abolished. Synthetic partially duplex oligonucleotide templates containing Ad origin sequences were constructed in order to determine the structure of the DNA molecules that are active in the absence of factor pL. These experiments indicated that factor pL degrades the 5'-end of the nontemplate (displaced) strand of the Ad origin thereby creating a single-stranded region at the 3'-end of the template strand. Such DNAs are competent for initiation of Ad DNA replication in the absence of factor pL but remain dependent on nuclear factor 1.

Adenoviridae↗

PubMed1986

[Coupling and rec-independence of the processes of replication and transposition of Pseudomonas aeruginosa phage D3112. The effect of the phage genes controlling the replication of DNA of D3112].

D3112 phage was shown to replicate via the process of coupled replication--transposition: the phage DNA is not excised from the chromosome after prophage induction and new phage copies insert into many different sites. The transposition is controlled by two D3112 early genes--A (mapped in the 1.5-3 kbp region) and B (3-4.5 kbp), and requires intact attL site (involvement of the phage right end attR not studied). D3112 is capable to transpose RP4 plasmid into the chromosome; both the D3112 and RP4 transpositions are rec-independent. The product of the early C gene which is not required for D3112 transposition has pleiotropic effect on the development of D3112 and is necessary for the process of D3112 DNA excision from the chromosome, for cell lysis as well as for mature phage production. We suggest that this gene is responsible for positive regulation of D3112 late genes expression, similar to the C gene of Mu phage or Q gene of lambda. Mutations in four D3112 late genes ts25, ts35, ts73 and ts110 do not affect transposition or excision processes. No detectable (less than 0.02 copies per cell) amount of linear or circular D3112 DNA is formed during the replication--transposition. Hence, in the course of replication and transposition processes D3112 genome has its ends permanently bound covalently to the chromosome. The excision of the D3112 DNA takes place at late stages.

Bacteriophages↗

PubMed1989

Replication of ColE2 and ColE3 plasmids: in vitro replication dependent on plasmid-coded proteins.

We developed an in vitro replication system for ColE2 and ColE3 plasmids using cell extracts prepared from bacteria with or without these plasmids. DNA synthesis depended on host DNA polymerase I and was sensitive to rifampicin and chloramphenicol. Preincubation of the extracts with plasmid DNA, however, allowed replication of template DNA added subsequently in a plasmid-specific manner in the presence of rifampicin and chloramphenicol. The plasmid-specified trans-acting factor(s) was detected in cell extracts from bacteria carrying a recombinant plasmid with the region of ColE2 or ColE3 encoding the Rep protein. The plasmid-specified factor(s) consisted at least in part of protein, probably the Rep protein. In vitro replication started within a region of ColE2 or ColE3 containing the smallest cis-acting segment essential for in vivo replication and proceeded in a fixed direction.

Bacterial Proteins↗

PubMed1979

Plasmid replication functions. II. Cloning analysis of the repA replication region of antibiotic resistance plasmid R6-5.

R6-5 is a low copy number, conjugative, FII incompatibility group plasmid that has a molecular length of 102 kb and that specifies resistance against several antibiotics (chloramphenicol, fusidic acid, kanamycin, streptomycin and sulphonamide) and mercury salts. By means of in vitro cloning procedures, mini plasmids have been generated that contain a DNA segment from the essential region of R6-5 that is only 2.6 kb in length. This DNA segment, which consists of two PstI fragments that are adjacent in the parent plasmid, carries all genes and sequences required for the regulated replication and incompatibility properties of R6-5, including its origin of replication, OriV, an essential function that has been designated RepA, and the copy control function, Cop. Three different polypeptides, having monomer molecular weights of 23,000, 10,000 and 9,500 daltons, are synthesized in detectable quantities by minicells carrying pBR322 hybrid plasmids that contain DNA segments from the R6-5 essential region. A spontaneous deletion derivative of a pBR322 hybrid plasmid that carries the R6-5 origin of replication was isolated. Heteroduplex analysis of this derivative plasmid indicates that the deleted DNA segment carries the R6-5 replication origin and that its termini consist of short inverted repeat sequences.

DNA Polymerase I↗

PubMed1989

Dam methylated and hemimethylated oriC plasmids are replicated symmetrically; a novel and general test of replication symmetry.

Deoxyadenosine methylation (dam) of the numerous GATC sequences present in the Escherichia coli origin of chromosomal replication (oriC) has been shown to be important both in vivo and in vitro for efficient initiation of DNA synthesis. Recent in vivo data suggest that initiation is only inefficient when these sequences are hemimethylated. This raises the interesting possibility that initiation may be inefficient because it only takes place on one strand of the template, i.e., replication is asymmetric on hemimethylated DNA. We tested this possibility by a novel and rapid approach which relies on the specificities of the restriction endonucleases MboI, MboII and DpnI. Although we show that replication takes place equally well on both strands of methylated and hemimethylated oriC DNA templates, the method should be applicable to the analysis of replication symmetry on most DNA templates which contain methylated deoxyadenosine GATC sequences as part of MboII restriction sites.

Base Sequence↗

PubMed1977

Semiconservative DNA replication in vitro. II. Replicative intermediates of mouse P-815 cells.

DNA chain growing during semiconservative replication was studied using both in vitro systems described in the preceding paper (preceding paper, ref 1) 3H-Labeled, 4-S Okazaki fragments synthesized in vivo just prior to permeabilization or lysis with Brij-58 were metabolically stable and quantitatively chased into high molecular weight DNA (20--100 S) during a subsequent incubation in vitro. Thus, DNA replication continued in vitro at the same growing points that were active in vivo. After a 20-s pulse at 30 degress C in vitro, more than 50% of incorporated radioactivity was found in the 4 S region of alkaline sucrose gradients suggesting a totally discontinuous mode of DNA chain growth. If the pulse were followed by a 1-min chase, 4-S molecules were converted into 6--12-S intermediates which upon continued incubation were joined with growing 20--100-S molecules (replicon-sized chains). Formation of all three classes of replicative intermediates, Okazaki fragments, 6--12-S intermediates, and 20--100-S molecules, occurred in vitro at least during the first 20 min. During this time, average rates of DNA chain growth and overall DNA synthesis were reduced to about the same extent, if compared to rates of intact cells. Thus, reduced chain growth rates appear to reflect primary deficiences of our in vitro systems, while initiation of replicative intermediates still occurs.

Animals↗

PubMed1988

Initiation of lambda DNA replication reconstituted with purified lambda and Escherichia coli replication proteins.

Using highly purified bacteriophage lambda and E. coli replication proteins, we were able to reconstitute an in vitro system capable of replication ori lambda-containing plasmid DNA. The addition of a new E. coli factor, the grpE gene product, to this replication system reduced the level of dnaK protein required for efficient DNA synthesis by at least 10-fold, and also allowed the isolation of a stable DNA replication intermediate. Based on all available information, we propose a molecular mechanism for the action of the dnaK and grpE proteins during the prepriming reaction leading to lambda DNA synthesis.

Bacterial Proteins↗

PubMed1975

Reconstruction of bacteriophage T4 DNA replication apparatus from purified components: rolling circle replication following de novo chain initiation on a single-stranded circular DNA template.

The protein products of T4 bacteriophage genes 41, 43, 45, 44, and 62 have been purified to near homogeneity using an assay which measures their stimulation of DNA synthesis in a crude lysate of Escherichia coli cells in fected by an appropriate mutant phage. When all of these proteins and T4 gene 32 protein are incubated in the presence of deoxyribonucleoside and ribonucleoside triphosphates, extensive DNA synthesis occurs on both single and double-stranded DNA templates. Analysis of this in vitro system reveals most of the features attributed to in vivo DNA replication: (1) De novo DNA chain initiation is found on a single-stranded DNA template only if ribonucleoside triphosphates are present (as expected for RNA priming of Okazaki pieces on the "lagging" strand of a replication fork). (2) With single-stranded circular DNA as template, synthesis continues for many doublings. The products after extensive synthesis resemble a rolling circle as visualized in the electron microscope, with discontinuous "lagging" strand synthesis generating a long, unbranched double-stranded tail. The fact that all six mutationally identified T4 replication gene products are required for these syntheses suggests the existence of a large multienzyme complex, constituting the T4 replication apparatus.

Chromosome Mapping↗

PubMed1987

Host control of plasmid replication: requirement for the sigma factor sigma 32 in transcription of mini-F replication initiator gene.

Replication of F factor or mini-F plasmid is strongly inhibited in the rpoH (htpR) mutants of Escherichia coli deficient in the sigma factor (sigma 32) known to be required for heat shock gene expression. Transcription of the mini-F repE gene encoding a replication initiator protein (E protein) was examined by operon fusion and by direct determination of repE mRNA. The synthesis rate and the level of repE mRNA were found to increase transiently upon temperature upshift (30 degrees C to 42 degrees C) in wild-type cells but to decrease rapidly in the rpoH mutants. Thus sigma 32 appeared to be directly involved in transcription of repE whose product, E protein, in turn activates DNA replication from the mini-F ori2 region. This scheme of host-controlled plasmid replication is further supported by the analysis of transcription in vitro: RNA synthesis can be initiated from the repE promoter by a minor form of RNA polymerase containing sigma 32 but not by the major polymerase containing the normal sigma factor sigma 70. The sigma 32-mediated transcription from the repE promoter is strongly inhibited by the E protein. We conclude that transcription of the mini-F repE gene is mediated by the host transcription factor sigma 32 and is negatively controlled by its own product.

Bacterial Proteins↗

PubMed1976

DNA replication and post-replication repair in U.V.-sensitive mouse neuroblastoma cells.

Mouse neuroblastoma cells differentiate when grown in the absence of serum; differentiation is reversed on the addition of serum. Differentiated cells are more sensitive to U.V.-radiation than proliferating cells. Whereas addition of serum to differentiated neuroblastoma cells normally results in immediate, synchronous entry into S phase, irradiation just before the addition of serum results in a long delay in the onset of DNA replication. During this lag period, incorporated 3H-thymidine appears in the light density region of CsCl gradientss, reflecting either repair synthesis or abortive replication. Post-replication repair (gap-filling) was found to be present in proliferating cells and at certain times in differentiated cells. It is suggested that the sensitivity of differentiated neuroblastoma cells to U.V.-radiation may be due to ineffective post-replication repair or to deficiencies in more than one repair mechanism, with reduction in repair capacity beyond a critical threshold.

Cell Differentiation↗

PubMed1990

Human immunodeficiency virus type 1 (HIV-1) and herpes simplex virus type 2 (HSV-2) can coinfect and simultaneously replicate in the same human CD4+ cell: effect of coinfection on infectious HSV-2 and HIV-1 replication.

Experiments were designed to determine whether HIV-1 and herpes simplex virus type 2 (HSV-2) coinfection leads to simultaneous replication of both viruses in the same human CD4+ cell (MT-4 cell line) and the possible effects of coinfection on infectious virus production. Results from transmission electron microscopy analysis revealed replication of typical HSV-2 nucleocapsids in the nucleus and budding of HIV-1 particles through the plasma membrane and through intracytoplasmic vacuoles containing enveloped HSV-2 particles in the same coinfected cell. Coinfection of HIV-1 persistently infected H9IIIB or promonocytic U1 cells with HSV-2 did not alter total production of infectious HSV-2 or the percentage of HSV-2 infectious centers compared with control H9 and U937 cells infected with HSV-2 alone. However, in coinfected promonocytic U1 cells HSV-2 induced infectious HIV-1 production measured by syncytial plaque assay. In summary, both HIV-1 and HSV-2 can coinfect and simultaneously replicate in the same human CD4+ cell. Interactions between HIV-1 and HSV-2 appear to be unidirectional, resulting in accelerated replication of HIV-1 as reported by Albrecht et al. (J Virol 1989;63:1861-1868), but not HSV-2 as shown by us.

Acquired Immunodeficiency Syndrome↗

PubMed1988

Phospholipid modulates in vitro replication of autonomous replicating sequence from human cells.

A cloned plasmid, pmyc(H-K), containing sequences derived from human c-myc gene replicated in vitro in Raji nuclear extract in a semiconservative manner. Using this system, it was found that phosphatidylinositol and cardiolipin strongly inhibited the replication of pmyc(H-K) in vitro, whereas other phospholipids, i.e., phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, and sphingomyelin, had no appreciable effect. The concentrations of phosphatidylinositol and cardiolipin producing 50% inhibition of the replication were 4.6 and 5.4 microM, respectively. Phosphatidylinositol and cardiolipin inhibited the relaxation of pmyc(H-K) supercoiled DNA, but showed little or weaker effects on DNA polymerase alpha and topoisomerase II in Raji nuclear extract. These results suggest that phosphatidylinositol and cardiolipin antagonize the replication of pmyc(H-K) in vitro, through, at least in part, the interaction with topoisomerase I.

DNA Replication↗

PubMed1989

Overinitiation of replication of the Escherichia coli chromosome from an integrated runaway-replication derivative of plasmid R1.

A 16-base-pair fragment, deletion of which completely inactivated oriC, was replaced by a temperature-dependent runaway-replication derivative (the copy number of which increases with temperature) of the IncFII plasmid R1. The constructed strains were temperature sensitive, and flow cytometry revealed a severalfold increase in the DNA/mass ratio following shifts to nonpermissive temperatures. The cell size distribution was broader in the constructed strains relative to that in the wild type because of asynchrony between the chromosome replication and cell division cycles. This difference was more pronounced for counterclockwise initiation of chromosomal replication, in which small DNA-less cells and long filaments were abundant. Following a temperature shift the cell size distributions became even more broad, showing that changes in the frequency of chromosomal replication affect cell division and emphasizing the interplay between these two processes.

Chromosomes, Bacterial↗

PubMed1989

Escherichia coli dnaT gene function is required for pBR322 plasmid replication but not for R1 plasmid replication.

Plasmid pBR322 was unable to replicate in a temperature-sensitive dnaT1 strain at a nonpermissive temperature, whereas a pBR322-derived plasmid carrying the wild-type dnaT+ gene was able to replicate under the same conditions. In contrast to pBR322, plasmid R1 could replicate in the dnaT1 strain at a nonpermissive temperature. In keeping with this finding, in vitro replication of plasmid R1 did not require DnaT protein.

DNA Replication↗