Replication Of Replicative Form DNA Research Articles

40] φX174-Type primosomal proteins: Purification and assay

Publisher Summary This chapter describes the preparation of crude soluble extracts that are capable of supporting φX174 ss(c) → RF DNA replication. It is a complementation assay used to score the activity of some of the replication proteins. It is also used to score the purification of the primosomal proteins from Escherichia coli strains engineered to overproduce them, and the reconstitution of φX174 ss(c)→RF DNA replication with purified proteins. Resolution and reconstitution of the enzymatic activities required for conversion in vitro of φX174 single-stranded (circular) DNA [ss(c)] to the double-stranded replicative form (RF) have resulted in the identification of the two major cellular replication machines, the DNA polymerase III holoenzyme (DNA Pol III HE) and the primosome. The primosome contributes the priming and DNA helicase functions at the replication fork. Seven proteins are required for primosome assembly: DnaB, DnaC, DnaG, DnaT, PriA, PriB, and PriC. Assembly occurs at a specific sequence [a primosome assembly site (PAS) 8] on φX174 ss(c) DNA coated with the single-stranded DNA binding protein (SSB). Once assembled, the primosome can both translocate in either direction along the ssDNA and act as a DNA helicase in either direction. 3 The 3' → 5' movement and helicase activity is fueled by the ATPase (or dATPase) activity of PriA, whereas 5' → 3' movement and helicase activity is fueled by the NTPase activity of DnaB. Primase (DnaG) does not remain stably associated with the primosome. When associated with the preprimosome, forming the primosome, primase catalyzes the synthesis of RNA or DNA primers utilized by the DNA Pol III HE to initiate nascent strand synthesis.

Properties of the A and A proteins of bacteriophage G4. The origin of G4 replicative-form DNA replication.

The A and A proteins of the bacteriophage G4 have been purified. The proteins have been analysed for their enzymatic activities on single-stranded and double-stranded DNA. The A protein introduces a single-stranded break at a specific place in the G4 replicative form I DNA. This cleavage site has been localized between nucleotides 506 and 507 in the viral (+) strand. The A protein binds covalently to the 5' end of the cleavage site. The A protein initiates the replication of the viral (+) DNA [Borrias, et al. (1979) Virology, 31, 288-298]; the cleavage site therefore identifies the origin of replication. The A protein cleaves viral (+) strand DNA at many different sites and also binds covalently to the 5' ends of the nick sites. The properties of both proteins strongly resemble the properties of the A and A proteins of the related and much butter analysed phage phi X174. These results indicate that the G4 and phi X174A and A proteins have comparable functions and also that both phages initiate the replicative form DNA in a similar way.

Identification of a ØX174 coded protein involved in the inhibit on of β-galactosidase synthesis in [formula omitted

The synthesis of β-galactosidase (EC 3.2.1.23: β-D-galactoside galactohydrolase) in Escherichia coli is repressed as a result of infection with single-stranded DNA phage ØX174. An amber mutant in ØX174 cistron A, which codes for two proteins, does not inhibit the enzyme synthesis while amber mutants in all other genes do cause repression. A mutant near the amino-terminal end of cistron A, which produces the small 35,000 molecular weight cistron A polypeptide, also inhibits the synthesis of β-galactosidase. Inhibition is also observed in an Escherichia coli rep mutant which does not support the replication of replicative-form DNA. Exogenous nucleotide bases and cyclic 3′,5′-adenosine monophosphate (cyclic AMP) do not have any effect on the degree of repression.

Bacteriophage φX174 and G4 RF DNA replicative intermediates: A comparative study using different isolation procedures

We have isolated replicative intermediates of bacteriophage φX174 and the related baeteriophage G4, during RF (replicative form) DNA replication using different procedures. Biochemical and electron microscopic analysis of φX and G4 DNA replicative intermediates isolated by the same procedure, showed no significant differences. In the replication cycle of both phages rolling circles and gapped RF DNA molecules are the predominant replicative intermediates. It is concluded that G4 RF DNA also replicates according to a rolling circle model and not according to a D-looped replication model as proposed by Godson (1977 b).

Replication of bacteriophage phiK duplex replicative-form DNA in dnaB and dnaC mutants of Escherichia coli

We have directly tested the effects of host cell DNA synthesis mutations on bacteriophage phiK replicative-form (RF) DNA replication in vivo. We observed that phiK RF DNA replication continued at normal rates in both dnaB and dnaC mutant hosts under conditions in which the activities of the dnaB and dnaC gene products were shown to be markedly reduced. This suggests that these two host proteins are not essential for normal phiK RF DNA replication. In control experiments we observed markedly reduced rates of phiK RF DNA replication in temperature-sensitive dnaG and dnaE host mutants, indicating that the products of these genes are essential. Thus, the mechanism of DNA chain initiation in vivo on the duplex RF DNA templates of isometric phages such as phiK apparently is different from that on the similar templates of isometric phages such as phiX174. The implications of this difference are discussed in the text.

Cleavage Site of X174 Gene-A Protein in X and G4 RFI DNA

The circular, single-stranded (SS) DNA of bacteriophage ϕX174 is replicated via a double-stranded (DS) replicative form (RF) (Sinsheimer et al. 1962). The replication of ϕX RF DNA has been intensively studied from the viewpoint of clarifying the initiation mechanism of DNA synthesis at the molecular level. ϕX RF DNA replication is under the control of viral gene A, as was first shown by Tessman (1966) for the closely related bacteriophage S13. ϕX RF DNA replication is initiated by the introduction of a single nick in the viral strand of the superhelical, covalently closed, circular, duplex (RFI) DNA which is mediated by the viral gene-A protein (Francke and Ray 1971, 1972). The nick in the viral strand resides at a unique site on the ϕX genome which, surprisingly, is located within viral gene A as shown by in vivo (Baas et al. 1976) and in vitro studies (Ikeda et al. 1976;...

Bacteriophage phiX174 RF DNA replication in vivo: a biochemical study.

Abstract We have studied the replication of bacteriophage φX174 replicative form (RF) DNA by biochemical analysis of pulse-labelled replicative intermediates and covalently closed RFI † DNA. The pulse-labelled DNA was isolated and separated into fractions which were shown to contain rolling circles, partially double-stranded molecules, RFI and RFII molecules, respectively. In these molecules the position of the pulse label and its strand specificity was determined. The results obtained were strongly dependent on the method used to stop the pulse-labelling. When the pulse is ended by poison (KCN and NaN 3 ) and fast cooling significantly more label is found in the viral than in the complementary strand. In agreement with the rolling circle model, the majority of the viral strand label is present in linear genome length and longer than genome length φX DNA molecules. The radioactivity in the RFII fraction is found almost exclusively in linear viral φX DNA of genome length. Restriction enzyme analysis of this fraction indicates that the φX viral strand DNA synthesis terminates in the Hae III restriction fragment Z 6B . In contrast, the main part of the labelled complementary strand φX DNA is found in short pieces. Full length, pulse-labelled complementary strands are present in the RFI fraction and to a minor extent in the RFII fraction. Restriction enzyme analysis of full length complementary strands, from RFI and RFII, shows that there is no unique termination site for the complementary strand DNA synthesis. Analysis of the pulse-labelled covalently closed RFI fraction indicates that this fraction contains a collection of molecules with increasing superhelix density, ranging from relaxed RFI up to fully super-twisted RFI DNA. This suggests that at the end of a replication cycle the conversion of RFII into superhelical RFI DNA involves the formation of relaxed RFI DNA and the successive introduction of superhelical turns. When the pulse is ended by pouring the infected culture into a phenol/ethanol solution less radioactivity is found to be incorporated into φX DNA. Furthermore, the distribution of the label between the complementary and viral strand is more equal, and significantly more radioactivity is found in short pieces. A comparison of both stopping methods indicates that fast cooling in poison does not stop efficiently φX. RF DNA replication, especially the viral strand DNA synthesis and DNA ligase action.

Defective interfering particles of parvovirus H-1.

Defective interfering particles of the parvovirus H-1 were produced by serial propagation at high multiplicities of infection. Such particles interfere with the synthesis of capsid proteins and infectious virus of standard H-1. The interference is sensitive to UV irradiation, dependent on the multiplicity of the challenge virus, and is active in heterotypic infections against parvovirus H-3 or LuIII. Defective interfering particle genomes have alterations characterized by integral numbers (1 to 10 or more) of a 60-base-pair addition in the neighborhood of the origin of replicative-form DNA replication and deletions that are located primarily within two regions, 32 to 44 or 80 to 90 on the genome map. Some of the implications of these findings are discussed.

Replication process of the parvovirus H-1. X. Isolation of a mutant defective in replicative-form DNA replication.

A temperature-sensitive mutant of H-1, ts14, that is partially defective in replicative-form (RF) DNA synthesis has been isolated. ts14 H-1 is characterized by a decrease in plaque-forming ability and production of infectious virus at the restrictive temperature of 39.5 degrees C. RF DNA synthesis of ts14 is reduced to 3 to 7% of that of wild-type H-1 at either the restrictive or the permissive temperature. A complementation analysis of RF synthesis of ts14 and a viable defective H-1 virus, DI-1, or wild-type H-3 indicates that the defective RF DNA synthesis of ts14 is cis-acting. ts14, unlike wild-type H-1, causes a multiplicity-dependent inhibition of DI-1 or H-3, but not LuIII, RF DNA synthesis. Mixed infections of cells with two parvoviruses also exhibited a cross-interference for viral protein synthesis that was multiplicity dependent, ts14 inhibited infectious virus production of H-1 or H-3, but not LuIII. LuIII-or H-3-pseudotype particles were produced by coinfection with H-1. H-3 and H-1 showed similar interactions with ts14, and H-3 DNA was more homologous to H-1 than was LuIII by comparative physical mapping studies. The results suggest that ts14 is a mutant with a defect in a regulatory sequence of its DNA that influence RF DNA replication.

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).

Inhibition of bacteriophage .PHI.X174 replicative-form DNA replication by rifampicin.

ϕX174 DNA synthesis as well as phage production was inhibited by rifampicin when added in early phase of infection. Rifampicin did not inhibit the formation of parental duplex replicative-form, RF, and it inhibited the synthesis of progeny RF under conditions where protein synthesis was not necessary to be synthesized continuously. In addition, replication of parental RF into progeny RF was inhibited by rifampicin under conditions where a high concentration of chloramphenicol did not affect the replication. Consequently, it could be concluded that RNA synthesis other than that required for protein synthesis was necessary for both the initiation and continuation of RF replication.

Bacteriophage G4 DNA synthesis in temperature-sensitive dna mutants of Escherichia coli.

The synthesis of bacteriophage G4 DNA was examined in temperature-sensitive dna mutants under permissive and nonpermissive conditions. The infecting single-stranded G4 DNA was converted to the parental replicative form (RF) at the nonpermissive temperature in infected cells containing a temperature sensitive mutation in the dnaA, dnaB, dnaC, dnaE, or dnaG gene. The presence of 30 mug of chloramphenicol or 200 mug of rifampin per ml had no effect on parental RF synthesis in these mutants. Replication of G4 double-stranded RF DNA occurred at a normal rate in dnaAts cells at the nonpermissive temperature, but the rate was greatly reduced in cells containing a temperature-sensitive mutation in the dnaB, dnaC, dnaE, or dnaG gene. RF DNA replicated at normal rates in revertants of these dna temperature-sensitive host cells. The simplest interpretation of these observations is that none of the dna gene products tested is essential for the synthesis of the complementary DNA strand on the infecting single-stranded G4 DNA, whereas the dnaB, dnaC, dnaE, (DNA polymerase III), and dnaG gene products are all essential for replication of the double-stranded G4 RF DNA. The alternate possibility that one or more of the gene products are actually essential for G4 parental RF synthesis, even though this synthesis is not defective in the mutant hosts, is also discussed.

Process of infection with bacteriophage phi X 174 XXXVIII. Replication of phi chi 174 replicative form in vivo.

The replication of bacteriophage phi X 174 replicative-form DNA has been studied by structural analysis of pulse-labeled replicative-intermediate molecules. Such intermediates were identified by pulse-labeling with [13H]thymidine and separated into four major fractions (A, B, C, and D) in a propidium diiodide-cesium chloride buoyand density gradient. Sedimentation analysis of each of these fractions suggests the following features of phi X replicative-form DNA replication in vivo. (i) At the end of one cycle of replication, one daughter replicative form (RFII) contains a nascent plus (+) strand of the unit viral length, and the other daughter RFII contains small fragments of nascent minus (-) strand. (ii) Asymmetry is also associated with production of the first supercoiled RFI after addition of pulse label in that only the minus strand becomes radioactive. (iii) A supercoiled DNA (RFI') seems to occur in vivo. This DNA is observed at a position of greater density in a propidium diiodide-cesium chloride buoyant density gradient than normal RFI. (iv) A novel DNA component is observed, at a density greater than RFI, which releases, in alkali, a plus strand longer (1.5 to 1.7 times) than the unit viral length. These results are discussed in terms of the possible sequence of events in phi X 174 replicative-form replication in vivo.

Synthesis of complex forms of bacteriophage phiX174 double-stranded DNA in a temperature-sensitive dnaC mutant of Escherichia coli C.

Fast-sedimenting forms of bacteriophage phiX174 double-stranded replicative-form DNA observed in normal infections continued to accumulate at the nonpermissive temperature in a temperature-sensitive dnaC mutant of Escherichia coli. These complex molecules accounted for up to half of the DNA synthesized during short pulses at the nonpermissive temperature. They were the dead-end products of DNA synthesis, not intermediates in normal replicative-form replication. The data suggest that these higher-than-normal-molecular-weight DNA molecules result from abnormal initiation of phiX174 replicative-form DNA replication.

Replication process of the parvovirus H-1. III. Factors affecting H-1 RF DNA synthesis.

Replication of the single-stranded DNA parvovirus H-1 involves the synthesis of a double-stranded DNA replicative form (RF). In this study, the metabolism of RF DNA was examined in parasynchronous hamster embryo cells. The initiation of RF DNA replication was found to occur late in S phase, as was the synthesis of the DNA upon which subsequent viral hemagglutinin synthesis is dependent. Evidence is presented which indicates that initiation of RF replication requires proteins synthesized in late S phase, but that concomittant protein synthesis is not required for the continuation of RF replication. The data also suggest a requirement for viral protein(s) for progeny strand synthesis. Incorporation of 5-bromo-2'-deoxyuridine (BUdR) into viral DNA resulted in an "all-or-none" inhibition of viral hemagglutinin and viral antigen synthesis. BUdR inactivation of viral protein function was used to explore the time of synthesis of viral DNA serving as template for viral RNA synthesis and the effect of viral protein on RF replication and progeny strand synthesis. Results of this study suggest that parental RF DNA is synthesized shortly after infection, and that viral mRNA is transcribed from only a few copies of the viral genome in each cell. They also support the conclusion that viral protein is inhibitory to RF DNA replication. Density labeling of RF DNA with BUdR, allowing separation of viral strand DNA (V) from viral complementary strand (C), provided additional data in support of the above findings.