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.     

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.     

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.     

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.     

Bacteriophage PRD1 DNA entry uses a viral membrane-associated transglycosylase activity

Amino acid sequence analyses have indicated that the amino-terminal part of bacteriophage PRD1 structural protein P7 carries a conserved transglycosylase domain. We analysed wild-type PRD1 and different mutant particles in zymograms and found a glycolytic activity that was associated with protein P7. This is the first time a putative bacteriophage or plasmid lytic transglycosylase has been shown to have an enzymatic activity. In the absence of protein P7, the phage DNA replication and host cell lysis were delayed. Gene VII of PRD1 is known to encode proteins P7 and P14. In this investigation, the open reading frame coding for P14 was mapped to the 3' end of gene VII. Proteins P7 and P14 probably form a heteromultimeric complex, which is located at the particle vertices and is involved in the early steps of the PRD1 life cycle     

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.     

Overexpression, purification, and characterization of Escherichia coli bacteriophage PRD1 DNA polymerase. In vitro synthesis of full-length PRD1 DNA with purified proteins.

The bacteriophage PRD1 DNA polymerase gene (gene I) has been cloned into the expression vector pPLH101 under the control of the lambda pL promoter. Tailoring of an efficient ribosome binding site in front of the gene by polymerase chain reaction led to a high level heat-inducible expression of the corresponding gene product (P1) in Escherichia coli cells. Expression was confirmed in vivo by complementation of phage PRD1 DNA polymerase gene mutants and in vitro by formation of the genome terminal protein P8-dGMP replication initiation complex. Expressed PRD1 DNA polymerase was purified to apparent homogeneity in an active form. DNA polymerase, 3'-5'-exonuclease, and P8-dGMP replication initiation complex formation activities cosedimented in glycerol gradient with a protein of 65 kDa, the size expected for PRD1 DNA polymerase. The DNA polymerase was active on DNase I-activated calf thymus DNA, poly(dA).oligo(dT) and poly(dA-dT) primer/templates as well as on native phage PRD1 genome. The 3'-5'-exonuclease activity was specific for single-stranded DNA and released mononucleotides. No 5'-3'-exonuclease activity was detected. The inhibitor/activator spectrum of the PRD1 DNA polymerase was also studied. An in vitro replication system with purified components for bacteriophage PRD1 was established. Formation of the P8-dGMP replication initiation complex was a prerequisite for phage DNA replication, which proceeded from the initiation complex and yielded genome length replication products.     

Protein—nucleic acid interactions in bacteriophage φ29 DNA replication

Abstract: φ29 DNA replication starts at both DNA ends by a protein priming mechanism. The formation of the terminal protein-dAMP initiation complex is directed by the second nucleotide from the 3' end of the template. The transition from protein-primed initiation to normal DNA elongation has been proposed to occur by a sliding-back mechanism that is necessary for maintaining the sequences at the φ29 DNA ends. Structure—function studies have been carried out in the φ29 DNA polymerase. By site-directed mutagenesis of amino acids conserved among distantly related DNA polymerases we have shown that the N-terminal domain of φ29 DNA polymerase contains the 3'–5' exonuclease activity and the strand-displacement capacity, whereas the C-terminal domain contains the synthetic activities (protein-primed initiation and DNA polymerization). Viral protein p6 stimulates the initiation of φ29 DNA replication. The structure of the protein p6—DNA complex has been determined, as well as the main signals at the φ29 DNA ends recognized by protein p6. The DNA binding domain of protein p6 has been studied. The results indicate that an α-helical structure located in the N-terminal region of protein p6 is involved in DNA binding through the minor groove. The φ29 protein p5 is the single-stranded DNA binding (SSB) protein involved in φ29 DNA replication, by binding to the displaced single-stranded DNA (ssDNA) in the replication intermediates. In addition, protein p5 is able to unwind duplex DNA. The properties of the φ29 SSB—ssDNA complex are described. Using the four viral proteins, terminal protein, DNA polymerase, protein p6 and the SSB protein, it was possible to amplify the 19285-bp φ29 DNA molecule by a factor of 4000 after 1 h of incubation at 30°C. The infectivity of the in vitro amplified DNA was identical to that of φ29 DNA obtained from virions.     

In vitro replication of bacteriophage PRD1 DNA. Metal activation of protein-primed initiation and DNA elongation.

Bacteriophage PRD1 replicates its DNA by means of a protein-primed replication mechanism. Compared to Mg2+, the use of Mn2+ as the metal activator of the phage DNA polymerase results in a great stimulation of the initiation reaction. The molecular basis of the observed stimulatory effect is an increase in the velocity of the reaction. The phage DNA polymerase is also able to catalyze the formation of the initiation complex in the absence of DNA template. Although the presence of Mn2+ does not affect either the polymerization activity or the processivity of the DNA polymerase, this metal is unable to activate the overall replication of the phage genome. This can be explained by a deleterious effect of Mn2+ on the 3'-5'-exonucleolytic and/or the strand-displacement activity, the latter being an intrinsic function of the viral DNA polymerase required for protein-primed DNA replication.     

Improved artificial origins for phage Φ29 terminal protein-primed replication. Insights into early replication events

The replication machinery of bacteriophage Φ29 is a paradigm for protein-primed replication and it holds great potential for applied purposes. To better understand the early replication events and to find improved origins for DNA amplification based on the Φ29 system, we have studied the end-structure of a double-stranded DNA replication origin. We have observed that the strength of the origin is determined by a combination of factors. The strongest origin (30-fold respect to wt) has the sequence CCC at the 3′ end of the template strand, AAA at the 5′ end of the non-template strand and 6 nucleotides as optimal unpairing at the end of the origin. We also show that the presence of a correctly positioned displaced strand is important because origins with 5′ or 3′ ssDNA regions have very low activity. Most of the effect of the improved origins takes place at the passage between the terminal protein-primed and the DNA-primed modes of replication by the DNA polymerase suggesting the existence of a thermodynamic barrier at that point. We suggest that the template and non-template strands of the origin and the TP/DNA polymerase complex form series of interactions that control the critical start of terminal protein-primed replication.     

In VitroProtein-primed Initiation of Pneumococcal Phage Cp-1 DNA Replication Occurs at the Third 3′ Nucleotide of the Linear Template: A Stepwise Sliding-back Mechanism

Phage Cp-1 fromStreptococcus pneumoniaemakes use of a protein-priming mechanism to start replication of its linear DNA: the first reaction consists of the addition of 5′ dAMP to a molecule of the primer protein, an initiation event occurring at both DNA ends. After elongation of the initiation complex, the primer protein remains linked to the 5′ end of the nascent DNA chain, and is subsequently referred to as terminal protein (TP). In this paper, using DNA-free extracts from Cp-1-infectedS. pneumoniae, we provide evidence that the formation of the covalent complex TP-dAMP is a template-instructed reaction and that ssDNA molecules can serve as templates for TP-primed replication. A mutational analysis of the 3′ terminal nucleotides of Cp-1 DNA reveals that a precise DNA sequence is required for efficient template recognition, and thatin vitroinitiation of Cp-1 DNA replication is directed by the third nucleotide of the template. However, the two terminal nucleotides are recovered during the first steps of elongation. A new variant of the sliding-back mechanism for protein-primed initiation, firstly described forBacillus subtilisphage φ29, is proposed to account for the maintenance of Cp-1 DNA ends. The results presented here reinforce the hypothesis that sliding-back must be a common feature in all genomes that use protein-priming to initiate replication.     

Involvement of phage phi29 DNA polymerase and terminal protein subdomains in conferring specificity during initiation of protein-primed DNA replication

To initiate ϕ29 DNA replication, the DNA polymerase has to form a complex with the homologous primer terminal protein (TP) that further recognizes the replication origins of the homologous TP-DNA placed at both ends of the linear genome. By means of chimerical proteins, constructed by swapping the priming domain of the related ϕ29 and GA-1 TPs, we show that DNA polymerase can form catalytically active heterodimers exclusively with that chimerical TP containing the N-terminal part of the homologous TP, suggesting that the interaction between the polymerase TPR-1 subdomain and the TP N-terminal part is the one mainly responsible for the specificity between both proteins. We also show that the TP N-terminal part assists the proper binding of the priming domain at the polymerase active site. Additionally, a chimerical ϕ29 DNA polymerase containing the GA-1 TPR-1 subdomain could use GA-1 TP, but only in the presence of ϕ29 TP-DNA as template, indicating that parental TP recognition is mainly accomplished by the DNA polymerase. The sequential events occurring during initiation of bacteriophage protein-primed DNA replication are proposed.     

The genome of lipid-containing bacteriophage PRD1, which infects gram-negative bacteria, contains long, inverted terminal repeats.

The bacteriophage PRD1 is a lipid-bearing phage that infects a wide variety of gram-negative bacteria, including Escherichia coli and Salmonella typhimurium when they contain the appropriate plasmid. It contains a linear duplex DNA molecule that is covalently bound by its 5' ends to a terminal protein. We report here that the PRD1 genome contains a 111-base-pair terminal inverted repeat which does not bear homology to that of any known linear duplex DNAs with terminal proteins. We further report that its 3' termini are susceptible to enzymatic digestion by exonuclease III.     

Identification of a protein bound to the termini of bacteriophage PRD1 DNA.

Lipid-containing bacteriophage PRD1 has a double-stranded DNA genome of about 14,500 nucleotide base pairs. The phage can infect Escherichia coli and Salmonella typhimurium as well as other gram-negative bacteria harboring an appropriate plasmid. [35S]methionine label is incorporated into the DNA band early in infection. The label remains associated with DNA through phenol extraction and boiling with sodium dodecyl sulfate. Nuclease treatment of the genome released a protein which migrated as an early phage-specific protein (P8). This protein is also necessary for phage DNA replication. By restriction enzyme analysis it was shown that protein was associated with the terminal restriction fragments. Extracts of infected cells catalyzed the labeling of protein P8 with [alpha-32P]dGTP.     

Translesion-synthesis DNA polymerases participate in replication of the telomeres in Streptomyces

Linear chromosomes and linear plasmids of Streptomyces are capped by terminal proteins that are covalently bound to the 5'-ends of DNA. Replication is initiated from an internal origin, which leaves single-stranded gaps at the 3'-ends. These gaps are patched by terminal protein-primed DNA synthesis. Streptomyces contain five DNA polymerases: one DNA polymerase I (Pol I), two DNA polymerases III (Pol III) and two DNA polymerases IV (Pol IV). Of these, one Pol III, DnaE1, is essential for replication, and Pol I is not required for end patching. In this study, we found the two Pol IVs (DinB1 and DinB2) to be involved in end patching. dinB1 and dinB2 could not be co-deleted from wild-type strains containing a linear chromosome, but could be co-deleted from mutant strains containing a circular chromosome. The resulting ΔdinB1 ΔdinB2 mutants supported replication of circular but not linear plasmids, and exhibited increased ultraviolet sensitivity and ultraviolet-induced mutagenesis. In contrast, the second Pol III, DnaE2, was not required for replication, end patching, or ultraviolet resistance and mutagenesis. All five polymerase genes are relatively syntenous in the Streptomyces chromosomes, including a 4-bp overlap between dnaE2 and dinB2. Phylogenetic analysis showed that the dinB1-dinB2 duplication occurred in a common actinobacterial ancestor.     

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.     

phi29 DNA polymerase residue Phe128 of the highly conserved (S/T)Lx(2)h motif is required for a stable and functional interaction with the terminal protein

Bacteriophage phi29 encodes a DNA-dependent DNA polymerase belonging to the eukaryotic-type (family B) subgroup of DNA polymerases that use a protein as primer for initiation of DNA replication. By multiple sequence alignments of DNA polymerases from such a family, we have been able to identify two amino acid residues specifically conserved in the protein-priming subgroup of DNA polymerases, a phenylalanine contained in the (S/T)Lx(2)h motif, and a glutamate belonging to the Exo III motif. Here, we have studied the functional role of these residues in reactions that are specific for DNA polymerases that use a protein-primed DNA replication mechanism, by site-directed mutagenesis in the corresponding amino acid residues, Phe128 and Glu161 of phi29 DNA polymerase. Mutations introduced at residue Phe128 severely impaired the protein-primed replication capacity of the polymerase, being the interaction with the terminal protein (TP) moderately (mutant F128A) or severely (mutant F128Y) diminished. As a consequence, very few initiation products were obtained, and essentially no transition products were detected. Interestingly, phi29 DNA polymerase mutant F128Y showed a decreased binding affinity for short template DNA molecules. These results, together with the high degree of conservation of Phe128 residue among protein-primed DNA polymerases, suggest a functional role for this amino acid residue in making contacts with the TP during the first steps of genome replication and with DNA in the further replication steps.     

Establishment of a physical and genetic map for bacteriophage PRD1

DNA was isolated from the lipid-containing bacteriophage PRD1 and subjected to restriction endonuclease analysis. The total genome size is 14.7 kb. PRD1 DNA was resistant to cutting by fifteen restriction endonucleases with six base specificity. HaeII made thirty-seven cuts in the DNA, MboI made one cut, and MnlI made six cuts. DNA that was not treated with protease yielded two fewer fragments when treated with HaeII. Evidence is presented to indicate that the PRD1 DNA has protein at the ends of the DNA. The thirty-eight HaeII fragments were ordered using the ladder technique of Smith and Birnstiel (1976) on MboI and MnlI fragments of the genome. Clones of HaeII partial digests of PRD1 DNA in pBR322 were analyzed by HaeII digestion and were then assigned to specific regions of the genome by their HaeII fragment composition. A comparison of the marker rescue characteristics of the cloned DNA with the overall restriction fragment map generated a physical map of the genome. Some genes that have not been mapped because of a lack of mutants or leakiness at restrictive conditions were mapped by studying the in vitro protein synthesis of restriction endonuclease fragments.