The complete nucleotide sequence of the left very early region of Escherichia coli bacteriophage PRD1 coding for the terminal protein and the DNA polymerase

DNA molecules replicating in a linear form have been found in certain viruses and plasmids of both prokaryotic and eukaryotic origin. Characteristic of this type of molecules are the proteins covalently linked to their 5' ends and inverted terminal nucleotide sequences. The molecules replicate via a protein-priming mechanism, where participants include terminal protein and a specific polymerase. We report here the nucleotide sequence of the left very early region of Escherichia coli bacteriophage PRD1. This region codes for the terminal protein and the phage DNA polymerase. The predicted amino acid sequence of the terminal protein does not share homology with those of other known terminal proteins. The PRD1 DNA polymerase shows four regions of extensive homology to that of Bacillus subtilis phage phi 29. One of these conserved regions is also found in several animal virus DNA polymerases.     

Mapping of the DNA linking tyrosine residue of the PRD1 terminal protein.

DNA replication of PRD1, a lipid-containing phage, is initiated by a protein-priming mechanism. The terminal protein encoded by gene 8 acts as a protein primer in DNA synthesis by forming an initiation complex with the 5'-terminal nucleotide, dGMP. The linkage between the terminal protein and the 5' terminal nucleotide is a tyrosylphosphodiester bond. The PRD1 terminal protein contains 13 tyrosine residues in a total of 259 amino acids. By site-directed mutagenesis of cloned PRD1 gene 8, we replaced 12 of the 13 tyrosine residues in the terminal protein with phenylalanine and the other tyrosine residue with asparagine. Functional analysis of these mutant terminal proteins suggested that tyrosine-190 is the linking amino acid that forms a covalent bond with dGMP. Cyanogen bromide cleavage studies also implicated tyrosine-190 as the DNA-linking amino acid residue of the PRD1 terminal protein. Our results further show that tyrosine residues at both the amino-terminal and the carboxyl-terminal regions are important for the initiation complex forming activity. Predicted secondary structures for the regions around the DNA linking amino acid residues were compared in three terminal proteins (phi 29, adenovirus-2, and PRD1). While the linking amino acids serine-232 (phi 29) and serine-577 (adenovirus-2) are found in beta-turns in hydrophilic regions, the linking tyrosine-190 of the PRD1 terminal protein is found in a beta-sheet in a hydrophobic region.     

In vitro replication of bacteriophage PRD1 DNA. Characterization of the protein-primed initiation site.

Bacteriophage PRD1 replicates its DNA by means of a protein-primed replication mechanism. Using single-stranded oligonucleotide templates carrying the sequence corresponding to the 25 first bases of the 3' end of PRD1 DNA, and Mg2+ as the activating metal ion of the phage DNA polymerase, we show that the fourth base from the 3' end of the template directs, by base complementarity, the dNMP to be linked to the phage terminal protein (TP) in the initiation reaction. This result suggests that phage PRD1 maintains its 3' end DNA sequences via a sliding-back mechanism. The single-stranded DNA templates could not be replicated by the PRD1 DNA polymerase, much in contrast to the natural TP-DNA. Nevertheless, the analysis of the transition products obtained with TP-DNA and origin-containing oligonucleotides suggests that sliding-back occurs stepwise, the fourth base being the directing position during the entire process.     

Primary structure of the DNA terminal protein of bacteriophage PRD1.

The genome of a lipid-containing phage, PRD1, is replicated by a protein-priming mechanism. We have determined the nucleotide sequence of the PRD1 gene 8 which specifies the terminal protein, the protein primer for DNA synthesis. The coding region is 780 base pairs long and encodes for 259 amino acids (29,326 daltons). The predicted amino acid sequence of the PRD1 terminal protein reveals no substantial homology with that of any known terminal protein. However, hydropathy profiles of the PRD1, phi 29, and Nf terminal proteins are remarkably similar, suggesting a common evolutionary origin. A particular tyrosine residue is predicted to be covalently linked to the 5' end of the PRD1 DNA. The initiation codon ATG of gene 8 is preceded by the identifiable ribosome binding site, and putative promoter sequences. There are unique palindromic sequences between the ribosome binding site and "-10" region.     

Sequence requirements for protein-primed DNA replication of bacteriophage PRD1

In vitro studies have demonstrated that linear duplex, protein-free DNA molecules containing an inverted terminal repeat (ITR) sequence of the PRD1 genome at one end can undergo replication by a protein-primed mechanism. No DNA replication was observed when the ITR sequence was deleted or was not exposed at the terminus of the template DNA. We have determined the minimal origin of replication by analyzing the template activity of various deletion derivatives. Our results showed that the terminal 20 base-pairs of ITR are required for efficient in vitro DNA replication. We have found that, within the minimal replication origin region, there are complementary sequences. A site-specific mutagenesis analysis showed that most of the point mutations in the complementary sequences markedly reduced the template activity. The analyses of the results obtained with synthetic oligonucleotides have revealed that the specificity of the replication origin is strand specific and even on a single-stranded template a particular DNA sequence including a 3'-terminal C residue is required for the initiation of PRD1 DNA replication in vitro.     

Probing the relationship between RNA-stimulated ATPase and helicase activities of HCV NS3 using 2'-O-methyl RNA substrates

The hepatitis C virus (HCV) NS3 protein contains an amino terminal protease (NS3 aa. 1-180) and a carboxyl terminal RNA helicase (NS3 aa. 181-631). NS3 functions as a heterodimer of NS3 and NS4A (NS3/4A). NS3 helicase, a nucleic acid stimulated ATPase, can unwind RNA, DNA, and RNA:DNA duplexes, provided that at least one strand of the duplex contains a single-stranded 3' overhang (this strand of the duplex is referred to as the 3' strand). We have used 2'-O-methyl RNA (MeRNA) substrates to study the mechanism of NS3 helicase activity and to probe the relationship between its helicase and RNA-stimulated ATPase activities. NS3/4A did not unwind double-stranded (ds) MeRNA. NS3/4A unwinds hybrid RNA:MeRNA duplex containing MeRNA as the 5' strand but not hybrid duplex containing MeRNA as the 3' strand. The helicase activity of NS3/4A was 50% inhibited by 40 nM single-stranded (ss) RNA but only 35% inhibited by 320 nM ss MeRNA. Double-stranded RNA was 17 times as effective as double-stranded MeRNA in inhibiting NS3/4A helicase activity, while the apparent affinity of NS3/4A for ds MeRNA differed from ds RNA by only 2.4-fold. However ss MeRNA stimulated NS3/4A ATPase activity similar to ss RNA. These results indicate that the helicase mechanism involves 3' to 5' procession of the NS3 helicase along the 3' strand and only weak association of the enzyme with the displaced 5' strand. Further, our findings show that maximum stimulation of NS3 ATPase activity by ss nucleic acid is not directly related to procession of the helicase along the 3' strand.     

Replication at the telomeres of the Streptomyces linear plasmid pSLA2

The Streptomyces linear plasmid pSLA2 initiates DNA replication bidirectionally towards its telomeres from a site located near the centre of the molecule; at the telomeres, the recessed ends of lagging strands are filled in by non-displacing DNA synthesis. Here, we report experiments that test three proposed mechanisms for lagging-strand fill-in. We present data inconsistent with recombinational or terminal hairpin models for the formation of full-length duplex pSLA2 DNA. Instead, we find that deletions in short, distantly separated homologous palindromes in the leading-strand 3' overhang prevent propagation of linear pSLA2 DNA, implicating a mechanism of palindrome-mediated leading-strand fold-back in telomere replication. We further show that circularized pSLA2 DNA molecules are opened in vivo precisely at the terminal nucleotides of telomeres, generating functional linear replicons containing native telomeres covalently bound to a protein at their 5' DNA termini. Together, our results support a model in which pairing of multiple widely separated pSLA2 palindromes anchors the 3' end of the leading-strand overhang to a site near the overhang's base — providing a recognition site for terminal-protein-primed DNA synthesis and subsequent endonucleolytic processing. Thus, the replication of Streptomyces plasmid telomeres may have features in common with the mechanism proposed for telomere replication in autonomous parvoviruses.     

Characteristics of PRD1, a Plasmid-Dependent Broad Host Range DNA Bacteriophage

Several distinctive properties of PRD1, an icosahedral plasmid-dependent phage, are described. The drug-resistance plasmid-dependent host range of PRD1 extends beyond the P incompatibility group and includes gram-negative bacteria containing plasmids of incompatibility groups N and W. PRD1 phage will infect pseudomonads and Enterobacteriaceae containing either a P or W incompatibility group plasmid. PRD1 adsorbs to the cell wall of R(+) bacteria and thus its infectivity indicates cell wall alterations by these drug-resistance plasmid groups. PRD1 nucleic acid is duplex DNA with an estimated molecular weight of 24 x 10(6). The appearance of PRD1 in electron micrographs is suggestive of lipid content in addition to its buoyant density of 1.348 in CsCl and its sensitivity to chloroform. The latent period of PRD1 varies with the R(+) host bacterial strain used for growth of the phage.     

Stability of 3' double nucleotide overhangs that model the 3' ends of siRNA

Thermodynamic parameters are reported for duplex formation in 1 M NaCl for 16 RNA sequences, each containing a core tetramer duplex, GGCC, and a 3' overhang consisting of two bases. The results indicate additional double-helical stability is conferred by the double 3' terminal overhang relative to the single 3' terminal overhang. A nearest-neighbor analysis of the data indicates that the free energy contribution at 37 degrees C of the second base in the double 3' terminal overhang varies from 0 to 0.7 kcal/mol. The second base in the 3' double overhang can contribute nearly the same stability to a duplex as a base pair or a 3' dangling overhang. Stability contribution of a dangling base, two nucleotides removed from the 3' end of a duplex, is dependent upon both the identity of the base as well as that of the dangling base that it neighbors. A second dangling base only increases the stability of the duplex when it is neighboring a 3' purine dangling nucleotide. Furthermore, a second dangling pyrimidine provides a greater contribution to duplex stability than a purine. A nearest-neighbor model was developed to predict the influence of 3' double overhang on the stability of duplex formation. The model improves the prediction of free energy and melting temperature when tested against six sequences with different core duplexes.     

The organization of the right-end early region of bacteriophage PRD1 genome

Bacteriophage PRD1 is the only protein-primed DNA replication system known to operate in Escherichia coli. The left-genome end of PRD1 contains the early genes for the terminal protein and the DNA polymerase. These genes have been sequenced and the proteins have been produced separately. In this investigation we completed the analysis of the PRD1 early DNA regions by cloning and sequencing the right end genome containing early genes XII and XIX. We compared the structure of the right- and left-terminal regions. The genome organization of both ends was found to be rather uniform. The inverted terminal repeats, the first promoters and the first translation start codons are located almost exactly at the same distance from the genome ends. The PRD1 early gene products, P12 and P19, do not share similarities with proteins found in other protein-primed replication systems.     

NMR structure of a 2',5' RNA favors A type duplex with compact C2'endo nucleotide repeat

In order to provide a structural basis for the unusual properties of 2',5' nucleic acids, especially their unsuitability as information molecules, we report here a high resolution NMR structure of a 2',5' RNA fragment r(GCCGCGGC). It forms an A type duplex with C2'endo compact nucleotide repeat, instead of the familiar C3'endo compact nucleotide (seen in RNA) supporting the deductions made earlier from stereochemical considerations. This data together with the observation that 2',5' nucleic acids require mandatory slide and displacement for duplex and triplex structure formation suggest their reluctance to form the biologically relevant B type duplex. It is argued that this lack of flexibility for helical polymorphism and other inadequacies as a consequence of this may be a contributing factor for the rejection of 2',5' links by nature. The structure exhibits interesting features such as the syn glycosyl conformation for the terminal guanine and a hydrogen bond between O3' hydroxyl and anionic oxygen of the phosphate.     

Purified Membrane-Containing Procapsids of Bacteriophage PRD1 Package the Viral Genome

Icosahedral-tailed double-stranded DNA (dsDNA) bacteriophages and herpesviruses translocate viral DNA into a preformed procapsid in an ATP-driven reaction by a packaging complex that operates at a portal vertex. A similar packaging system operates in the tailless dsDNA phage PRD1 (Tectiviridae family), except that there is an internal membrane vesicle in the procapsid. The unit-length linear dsDNA genome with covalently linked 5′-terminal proteins enters the procapsid through a unique vertex. Two small integral membrane proteins, P20 and P22, provide a conduit for DNA translocation. The packaging machinery also contains the packaging ATPase P9 and the packaging efficiency factor P6. Here we describe a method used to obtain purified packaging-competent PRD1 procapsids. The optimized in vitro packaging system allowed efficient packaging of defined DNA substrates. We determined that the genome terminal protein P8 is necessary for packaging and provided an estimation of the packaging rate.     

Initiation of bacteriophage PRD1 DNA replication on single-stranded templates

In vitro studies have demonstrated that single-stranded DNA molecules containing the 3' terminal nucleotides of the PRD1 DNA replication origin can support initiation by a protein-primed mechanism. We have determined the minimal requirements for priming by analyzing the template activity of various deletion derivatives. Our results showed that the 3'-terminal 15 nucleotides of the replication origin are sufficient for priming. The finding that the requirements for recognition of replication origin are different from those for priming suggests that there are two distinct steps during initiation of PRD1 DNA replication: first, recognition of the replication origin on double-stranded DNA and second, the priming event on single-stranded DNA. In addition our results showed that additional bases at the 3' end of templates did not affect priming activity, suggesting that the priming site is searched for from inside the template.     

In vitro resolution of covalently joined AAV chromosome ends

We have developed an assay for a key step in the replication of adeno-associated virus (AAV) DNA. We demonstrate the covalently joined ends of linear AAV DNA can be resolved in vitro to the open duplex configuration. Only extracts prepared from human cells that have been infected with both adenovirus and AAV are capable of carrying out the reaction. The reaction is initiated by a site-specific and strand-specific endonucleolytic cut at a terminal resolution site near the end of the AAV terminal palindrome. During resolution the orientation of the terminal palindrome is inverted, and the 3' viral strand is extended by DNA synthesis. The size of the newly synthesized 3' strand is nearly identical to that found in viral particles. These observations provide direct biochemical evidence for an essential step in the model for AAV DNA replication.     

Structural study of the lipid-containing bacteriophage PRD1 and its capsid and DNA components by laser Raman spectroscopy

The Raman spectrum of a virus contains the structural signature of each of its molecular components (Thomas, 1987). We report the first Raman spectrum obtained from an intact, lipid-containing virus--the icosahedral bacteriophage PRD1--and show that this spectrum contains characteristic structure markers for the major capsid protein, the packaged double-stranded DNA genome, and the viral membrane which resides between the capsid and DNA. We find that the packaged genome of PRD1 exhibits Raman markers typical of the B-DNA secondary structure. Comparison of the Raman spectrum of the packaged DNA with that of protein-free DNA extracted from the virion shows further that the B-form secondary structure is not significantly perturbed by packaging in the virion. The Raman signature of the PRD1 membrane, monitored within the virion at 4 degrees C, is that of a phospholipid liquid-crystalline phase. The PRD1 capsid, which comprises several hundred copies of the major coat protein P3 (product of viral gene III) and a few copies of minor proteins, incorporates P3 capsomers predominantly in the beta-sheet conformation. The beta-sheet structure of P3 is maintained in the fully assembled PRD1 virion, as well as in the empty capsid. The present results demonstrate the feasibility of obtaining structural information from the three different classes of biomolecules--nucleic acid, protein, and lipid--which constitute a membrane-lined virus particle. Our results also demonstrate that the coat protein and double-stranded DNA components of a lipid-containing bacteriophage share many structural features in common with bacteriophage lacking a lipid membrane.     

Moloney murine leukemia virus integration protein produced in yeast binds specifically to viral att sites.

The integration protein (IN) of Moloney murine leukemia virus (MuLV), purified after being produced in yeast cells, has been analyzed for its ability to bind its putative viral substrates, the att sites. An electrophoretic mobility shift assay revealed that the Moloney MuLV IN protein binds synthetic oligonucleotides containing att sequences, with specificity towards its cognate (MuLV) sequences. The terminal 13 base pairs, which are identical at both ends of viral DNA, are sufficient for binding if present at the ends of oligonucleotide duplexes in the same orientation as in linear viral DNA. However, only weak binding was observed when the same sequences were positioned within a substrate in a manner simulating att junctions in circular viral DNA with two long terminal repeats. Binding to att sites in oligonucleotides simulating linear viral DNA was dependent on the presence of the highly conserved CA residues preceding the site for 3' processing (an IN-dependent reaction that removes two nucleotides from the 3' ends of linear viral DNA); mutation of CA to TG abolished binding, and a CA to TA change reduced affinity by at least 20-fold. Removal of either the terminal two base pairs from both ends of the oligonucleotide duplex or the terminal two nucleotides from the 3' ends of each strand did not affect binding. The removal of three 3' terminal nucleotides, however, abolished binding, suggesting an essential role for the A residue immediately upstream of the 3' processing site in the binding reaction. These results help define the sequence requirements for att site recognition by IN, explain the conservation of the subterminal CA dinucleotide, and provide a simple assay for sequence-specific IN activity.     

Protein-primed DNA replication: role of inverted terminal repeats in the Escherichia coli bacteriophage PRD1 life cycle.

Escherichia coli bacteriophage PRD1 and its relatives contain linear double-stranded DNA genomes, the replication of which proceeds via a protein-primed mechanism. Characteristically, these molecules contain 5'-covalently bound terminal proteins and inverted terminal nucleotide sequences (inverted terminal repeats [ITRs]). The ITRs of each PRD1 phage species have evolved in parallel, suggesting communication between the molecule ends during the life cycle of these viruses. This process was studied by constructing chimeric PRD1 phage DNA molecules with dissimilar end sequences. These molecules were created by combining two closely related phage genomes (i) in vivo by homologous recombination and (ii) in vitro by ligation of appropriate DNA restriction fragments. The fate of the ITRs after propagation of single genomes was monitored by DNA sequence analysis. Recombinants created in vivo showed that phages with nonidentical genome termini are viable and relatively stable, and hybrid phages made in vitro verified this observation. However, genomes in which the dissimilar DNA termini had regained identical sequences were also detected. These observations are explained by a DNA replication model involving two not mutually exclusive pathways. The generality of this model in protein-primed DNA replication is discussed.