Viral Strand DNA Synthesis Research Articles

Identification of the initiation sequence for viral-strand DNA synthesis of wheat dwarf virus.

The intergenic region of the circular single-stranded DNA genome of geminiviruses contains a sequence potentially able to fold into a stem-loop structure. This sequence has been reported to be involved in viral replication by serving as the origin for rolling-circle replication. However, in wheat dwarf virus (WDV) a deletion of 128 bp, removing this sequence, surprisingly does not prevent de novo viral DNA synthesis, but instead abrogates the processing of replicative intermediates into monomeric genomes. This deletion mutant permitted us to study the initiation of viral-strand DNA synthesis independently from its termination and also to identify the sequence within which rolling-circle DNA replication of WDV begins. We have mapped the initiation site of replication to a pentanucleotide, TACCC, a sequence that occurs twice in the large intergenic region of WDV: it is found in the right half of the stem-loop sequence and again 170 bases upstream where it is part of a 15 nucleotide sequence highly homologous to the right half of the stem-loop sequence. Here we show that viral-strand DNA synthesis efficiently initiates at both sequences.

Construction and characterization of a phage-plasmid hybrid (phagemid), pCAK1, containing the replicative form of viruslike particle CAK1 isolated from Clostridium acetobutylicum NCIB 6444

A bacteriophage-plasmid hybrid (phagemid) designated pCAK1 was constructed by ligating 5-kbp Escherichia coli plasmid pAK102 (AprEmr) and the 6.6-kbp HaeIII-linearized replicative form of the CAK1 viruslike particle from Clostridium acetobutylicum NCIB 6444. Phagemid pCAK1 (11.6 kbp) replicated via the ColE1 replication origin derived from pAK102 in E. coli. Single-stranded DNA (ssDNA) molecules complexed with protein in a manner which protected ssDNA from nucleases were recovered from the supernatant of E. coli DH11S transformants containing pCAK1 in the absence of cell lysis. This suggests that the viral-strand DNA synthesis replication origin of CAK1 and associated gene expression are functional in E. coli DH11S. The single-stranded form of pCAK1 isolated from E. coli supernatant was transformed into E. coli DH5 alpha' or DH11S by electroporation. Isolation of ampicillin-resistant E. coli transformants following transformation suggests that the complementary-strand DNA synthesis replication origin of CAK1 is also functional in E. coli. The coat proteins associated with ssDNA of pCAK1 demonstrated sensitivity to proteinase K and various solvents (i.e., phenol and chloroform), similar to the results obtained previously with CAK1. Following phagemid construction in E. coli, pCAK1 was transformed into C. acetobutylicum ATCC 824 and C. perfringens 13 by intact cell electroporation. Restriction enzyme analysis of pCAK1 isolated from erythromycin-resistant transformants of both C. acetobutylicum and C. perfringens suggested that it was identical to that present in E. coli transformants.

Gene X of bacteriophage f1 is required for phage DNA synthesis: Mutagenesis of in-frame overlapping genes

The gene II protein of bacteriophage f1 is a site-specific endonuclease required for initiation of phage viral strand DNA synthesis. Within gene II is another gene, X, encoding a protein of unknown function identical to the C-terminal 27% of the gene II protein, and separately translated from codon 300 (AUG) of gene II. By oligonucleotide mutagenesis, we constructed phage mutants in which this codon has been changed to UAG (amber) or UUG (leucine), and propagated them on cells carrying a cloned copy of gene X on a plasmid. The amber mutant makes no gene X protein, and cannot grow in the absence of the complementing plasmid; the leucine-inserting mutant can make gene X protein, and grows normally without the plasmid. Without gene X protein, phage DNA synthesis (particularly viral strand synthesis) is impaired. We discuss this finding in the context of other known in-frame overlapping genes (particularly genes A and A ∗ of phage φX174), many of which are also involved in the specific initiation of DNA synthesis, and suggest applications for the mutagenic strategy we employed.

Enzymatic techniques for the isolation of random single-base substitutions in vitro at high frequency.

A general and efficient method has been developed to generate large numbers of single-base substitution mutations simply and rapidly. A unique f1 phage recombinant DNA cloning vector is described, which contains the phi X174 origin of viral strand DNA synthesis and allows one to direct mutagenesis to any specific segment of DNA. Gapped circular DNA is constructed by annealing viral single-stranded circular DNA [ss(c) DNA] with a mixture of linear duplex DNAs that have had their 3'-OH termini processively digested with Escherichia coli exonuclease III under conditions in which the resulting, newly generated 3'-OH termini present in the various hybrid molecules span the region of interest. Base changes are induced by misincorporation of an alpha-thiodeoxynucleoside triphosphate analog onto this primer-template, followed by DNA repair synthesis. The asymmetric segregation of mutants from wild-type sequences is accomplished by double-stranded replicative form DNA----ss(c) DNA synthesis in vitro, initiated from the phi X174 viral strand origin sequence present on the vector DNA. Mutated ss(c) DNA is screened by the dideoxy chain termination method. In one mutagenesis experiment, 21 independent single-base substitutions were isolated in a 72-nucleotide-long target region. DNA sequence analysis showed that all possible base transversions and transitions were represented.

DNA sequences which support activities of the bacteriophage phi X174 gene A protein.

The DNA sequence of 30 nucleotides which surrounds the origin of viral strand DNA replication is highly conserved amongst the icosahedral single-stranded DNA bacteriophages. The A gene of these phages encodes a protein which is required for initiation and termination of viral strand DNA synthesis and acts as a nicking-closing activity specifically within this 30-nucleotide sequence. A system of purified Escherichia coli host proteins and phi X174 gene A protein has been developed which specifically replicates in vitro the viral strand of phi X174 from RF (replicative form) I template DNA and yields single-stranded circular DNA products (RF leads to SS(c) DNA replication system). Recombinant plasmids carrying inserts derived from phage phi X174 or G4 DNA which range in length from 49 to 1175 base pairs and contain the 30-nucleotide conserved sequence have been shown to support phi X A protein-dependent DNA synthesis in vitro in this replication system. We report here that insertion of the 30-nucleotide sequence alone into pBR322 allows the resulting recombinant plasmids to support phi X A protein-dependent in vitro DNA synthesis as efficiently as phi X174 template DNA in the RF leads to SS(c) replication system. The 30-nucleotide sequence functions as a fully wild type DNA replication origin as determined by the rate of DNA synthesis and the structure of resulting DNA products. Furthermore, the DNA sequence requirements for nicking of RF I DNA by the phi X A protein and for supporting replication origin function have been partially separated. Homology to positions 1, 29, and 30 of the 30-nucleotide conserved sequence are not required for cleavage of RF I DNA by the A protein; homology to position 1 but not 29 or 30 is required for efficient DNA replication.

Mechanism of replication of bacteriophage phi X174 XIX. Initiation of phi X174 viral strand DNA synthesis at internal sites on the genome.

Bacteriophage phi X174 viral strand DNA molecules shorter than genome length found late in the infectious cycle in Escherichia coli were 5' end labeled with 32P. Hybridization of the 32P-labeled molecules to restriction enzyme fragments of phi X replicative form DNA revealed an excess of phi X molecules whose 5' ends mapped in HaeIII fragments Z3 and Z4 in comparison with fragments Z1 and Z2. This suggests that initiation of phi X174 viral strand DNA synthesis may occur at internal sites on the complementary strand. There are several appropriately located sequences that might serve as n' (factor Y) recognition sequences and thereby facilitate discontinuous synthesis of the viral strand.

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.

G4 DNA replication: II. Synthesis of viral progeny single-stranded DNA

Molecules with single-stranded tails (rolling circles) were isolated as replicating intermediates in G4 progeny single-stranded DNA synthesis. Lysates from infected cells harvested late in infection during single-stranded DNA synthesis were not deproteinised but analysed directly in caesium chloride and propidium diiodide gradients. The gradient fractionated them on the basis of tail length. If the lysates were first deproteinised however, the tailed replicative intermediates banded as a peak at a density just greater than that of replicative form II DNA (RFII) and did not spread down the gradient. The origin of synthesis of the viral strand tail was mapped by electron microscopy as 55 to 60% away from the single EcoRI cleavage site. Termination molecules finishing a round of viral strand DNA synthesis have been identified as molecules consisting of a closed single-stranded DNA circle attached by a very small region to the parent double-stranded DNA circle.

Bacteriophage φx174 DNA synthesis in a replication-deficient host: Determination of the origin of φX DNA replication

We have reinvestigated bacteriophage φX DNA synthesis in rep 3 − cells. In these cells adsorption, transcription and translation of φX occurs normally, but φX replicative form DNA replication is blocked. After infection, φX DNA was isolated and separated by propidium diiodide/CsCl gradients into RFI † † Abbreviations used: RFI, replicative form DNA with both strands closed; RFII, replicative form DNA with one or more discontinuities in either strand; CAM, chloramphenicol; m.o.i., multiplicity of infection; PrI 2, propidium diiodide. HaeIII and HindII, restriction endonucleases from Haemophilus aegyptius and Haemophilus influenzae Rd, respectively. Z and R, DNA fragments produced by HaeIII and HindII, respectively. and RFII, followed by chromatography on BND-cellulose. In addition to the results of Francke & Ray (1971, 1972), we found an increasing amount of viral strand DNA synthesis in each fraction as time of infection proceeds. The occurrence of viral strand DNA synthesis is dependent on the presence of a functional cistron A product. In the presence of 150 μg chloramphenicol/ml or in infections with am18 (gene A) no viral strand DNA synthesis could be detected. After completion of the parental RF molecule, [ 3H]thymidine enters almost exclusively into viral strand DNA. The greater part of this viral strand DNA is found in genome-length φX. DNA in both RFI and RFII. Alkaline sucrose sedimentation of the RFII indicates that post-infection viral DNA label is only in linear φX DNA. These results strongly suggest that an extensive nick translation of the viral strand of RFII, followed occasionally by a conversion to RFI, takes place after synthesis of a functional cistron A product. This conclusion could be confirmed by restriction enzyme analysis of φX RF DNA, isolated from rep 3 − cells infected with 32P-labeled phage in the presence of [ 3H]thymidine. With the restriction enzyme HaeIII a gradient in the 3H 32P ratio of the fragments was obtained extrapolating into the fragment Z 6B. From this gradient the origin of the φX DNA nick translation could be located in the left half of Z 6B at approximately 65% (185 base-pairs) from the Z 6A-Z 6B junction.