Φ29 DNA Research Articles

Disclosing the in vivo organization of a viral histone-like protein in Bacillus subtilis mediated by its capacity to recognize the viral genome

Organization of replicating prokaryotic genomes requires architectural elements that, similarly to eukaryotic systems, induce topological changes such as DNA supercoiling. Bacteriophage 29 protein p6 has been described as a histone-like protein that compacts the viral genome by forming a nucleoprotein complex and plays a key role in the initiation of protein-primed DNA replication. In this work, we analyze the subcellular localization of protein p6 by immunofluorescence microscopy and show that, at early infection stages, it localizes in a peripheral helix-like configuration. Later, at middle infection stages, protein p6 is recruited to the bacterial nucleoid. This migrating process is shown to depend on the synthesis of components of the 29 DNA replication machinery (i.e., terminal protein and DNA polymerase) needed for the replication of viral DNA, which is required to recruit the bulk of protein p6. Importantly, the double-stranded DNA-binding capacity of protein p6 is essential for its relocalization at the nucleoid. Altogether, the results disclose the in vivo organization of a viral histone-like protein in bacteria.

Functional linkages between replication proteins of genes 1, 3 and 5 of <i>Bacillus subtilis</i> phage <b>φ</b>29

Gene 1 product (gp1) of Bacillus subtilis phage φ29 has been shown to be involved in viral DNA replication in vivo, but the essential role is still unknown. As part of an ongoing effort to understand the role of gp1 in viral DNA replication, we investigated genetic interaction between gene 1 and other viral genes. Because φ29 mutants which do not produce functional gp1 show temperature-sensitive growth, we isolated temperature-resistant phages from the φ29 gene 1 mutants, and eventually, obtained nine extragenic suppressors. These suppressor mutations were located in two essential genes for φ29 DNA replication in vivo: gene 3 encoding terminal/primer protein (gp3) or gene 5 encoding viral single-stranded DNA binding protein (gp5). Most of these mutations resulted in single amino acid substitutions in the products. By trans-complementation assay, we confirmed that the absence of gp1 at non-permissive temperature can be compensated by the suppressors which have the single amino acid substitution in either gp5 or gp3. These results indicate that gp1 has functional relationship to gp5 and gp3. From the positions of amino acid substitutions in gp3, we propose its new regulatory subdomain at which other molecules including gp1 would interact with and regulate functions of gp3.

Terminal protein-primed amplification of heterologous DNA with a minimal replication system based on phage Φ29

The DNA amplification performed by terminal protein-primed replication systems has not yet been developed for its general use to produce high amounts of DNA linked to terminal protein (TP). Here we present a method to amplify in vitro heterologous DNAs using the Φ29 DNA replication machinery and producing DNA with TP covalently attached to the 5' end. The amplification requires four Φ29 proteins, DNA polymerase, TP, single-stranded DNA binding protein and double-stranded DNA binding protein (p6). The DNA to be amplified is inserted between two sequences that are the Φ29 DNA replication origins, consisting of 191 and 194bp from the left and right ends of the phage genome, respectively. The replication origins do not need to have TP covalently attached beforehand to be functional in amplification and they can be joined to the DNA to be amplified by cloning or ligation. The facts that two functional origins were required at the ends of a linear template DNA and that the kinetics of DNA synthesis was very similar to that obtained using the TP-containing Φ29 genome as template support the proposal that genuine amplification is taking place. Amplification factors of 30-fold have been obtained. Possible applications of DNAs produced by this method are discussed.

Whole genome sequencing of environmental Vibrio cholerae O1 from 10 nanograms of DNA using short reads

Multiple Displacement Amplification (MDA) of DNA using φ29 (phi29) DNA polymerase amplifies DNA several billion-fold, which has proved to be potentially very useful for evaluating genome information in a culture-independent manner. Whole genome sequencing using DNA from a single prokaryotic genome copy amplified by MDA has not yet been achieved due to the formation of chimeras and skewed amplification of genomic regions during the MDA step, which then precludes genome assembly. We have hereby addressed the issue by using 10ng of genomic Vibrio cholerae DNA extracted within an agarose plug to ensure circularity as a starting point for MDA and then sequencing the amplified yield using the SOLiD platform. We successfully managed to assemble the entire genome of V. cholerae strain LMA3984-4 (environmental O1 strain isolated in urban Amazonia) using a hybrid de novo assembly strategy. Using our method, only 178 out of 16,713 (1%) of contigs were not able to be inserted into either chromosome scaffold, and out of these 178, only 3 appeared to be chimeras. The other contigs seem to be the result of template-independent non-specific amplification during MDA, yielding spurious reads. Extraction of genomic DNA within an agarose plug in order to ensure circularity of the extracted genome might be key to minimizing amplification bias by MDA for WGS.

Whole-Genome Amplification Using Φ29 DNA Polymerase

The cornerstones of any genetic analysis study are the quality and quantity of the DNA samples. DNA is a precious limited resource, and in human disease studies the accessibility of sample DNA is often governed by the isolation method and the human source. Additionally, forensic analysis and archaeological research are generally infeasible without intact sample DNA. Therefore, mechanisms to preserve or enhance the quantity of the DNA stock are crucial to the success of these studies. Historically, to preserve and maintain DNA stocks, costly and labor-intensive Epstein-Barr-virus-transformed cell lines were produced. The creation of cell lines can be valuable for a number of reasons in addition to creating a renewable resource of DNA, but the cost and effort to create them, as well as the requirement of intact cells to begin with, limit the utility of this approach. More recently, whole-genome amplification (WGA), utilizing the unique property of the enzyme Φ29 DNA polymerase, has been used to generate robust high-fidelity copies of the genome. As described in this protocol, WGA using Φ29 DNA polymerase allows unbiased representation of the genome via multiple-strand displacement, followed by rolling-circle amplification on random primers.

Isothermal DNA Amplification in Bioanalysis: Strategies and Applications

Isothermal DNA amplification is an alternative to PCR-based amplification for point-of-care diagnosis. Since the early 1990s, the approach has been refined into a simple, rapid and cost-effective tool by means of several distinct strategies. Input signals have been diversified from DNA to RNA, protein or small organic molecules by translating these signals into input DNA before amplification, thus allowing assays on various classes of biomolecules. In situ detection of single biomolecules has been achieved using an isothermal method, leveraging localized signal amplification in an intact specimen. A few pioneering studies to develop a homogenous isothermal protein assay have successfully translated structure-switching of a probe upon target binding into input DNA for isothermal amplification. In addition to the detection of specific targets, isothermal methods have made whole-genome amplification of single cells possible owing to the unbiased, linear nature of the amplification process as well as the large size of amplified products given by ϕ29 DNA polymerase. These applications have been devised with the four isothermal amplification strategies covered in this review: strand-displacement amplification, rolling circle amplification, helicase-dependent amplification and recombinase polymerase amplification.

Digital MDA for enumeration of total nucleic acid contamination

Multiple displacement amplification (MDA) is an isothermal, sequence-independent method for the amplification of high molecular weight DNA that is driven by ϕ29 DNA polymerase (DNAP). Here we report digital MDA (dMDA), an ultrasensitive method for quantifying nucleic acid fragments of unknown sequence. We use the new assay to show that our custom ϕ29 DNAP preparation is free of contamination at the limit of detection of the dMDA assay (1 contaminating molecule per assay microliter). Contamination in commercially available preparations is also investigated. The results of the dMDA assay provide strong evidence that the so-called ‘template-independent’ MDA background can be attributed to high-molecular weight contaminants and is not primer-derived in the commercial kits tested. dMDA is orders of magnitude more sensitive than PCR-based techniques for detection of microbial genomic DNA fragments and opens up new possibilities for the ultrasensitive quantification of DNA fragments in a wide variety of application areas using MDA chemistry and off-the-shelf hardware developed for digital PCR.

Improvement of φ29 DNA polymerase amplification performance by fusion of DNA binding motifs

Bacteriophage ϕ29 DNA polymerase is a unique enzyme endowed with two distinctive properties, high processivity and faithful polymerization coupled to strand displacement, that have led to the development of protocols to achieve isothermal amplification of limiting amounts of both circular plasmids and genomic DNA. To enhance the amplification efficiency of ϕ29 DNA polymerase, we have constructed chimerical DNA polymerases by fusing DNA binding domains to the C terminus of the polymerase. The results show that the addition of Helix-hairpin-Helix [(HhH)(2)] domains increases DNA binding of the hybrid polymerases without hindering their replication rate. In addition, the chimerical DNA polymerases display an improved and faithful multiply primed DNA amplification proficiency on both circular plasmids and genomic DNA and are unique ϕ29 DNA polymerase variants with enhanced amplification performance. The reported chimerical DNA polymerases will contribute to make ϕ29 DNA polymerase-based amplification technologies one of the most powerful tools for genomics.

Viral terminal protein directs early organization of phage DNA replication at the bacterial nucleoid

The mechanism leading to protein-primed DNA replication has been studied extensively in vitro. However, little is known about the in vivo organization of the proteins involved in this fundamental process. Here we show that the terminal proteins (TPs) of phages ϕ29 and PRD1, infecting the distantly related bacteria Bacillus subtilis and Escherichia coli, respectively, associate with the host bacterial nucleoid independently of other viral-encoded proteins. Analyses of phage ϕ29 revealed that the TP N-terminal domain (residues 1-73) possesses sequence-independent DNA-binding capacity and is responsible for its nucleoid association. Importantly, we show that in the absence of the TP N-terminal domain the efficiency of ϕ29 DNA replication is severely affected. Moreover, the TP recruits the phage DNA polymerase to the bacterial nucleoid, and both proteins later are redistributed to enlarged helix-like structures in an MreB cytoskeleton-dependent way. These data disclose a key function for the TP in vivo: organizing the early viral DNA replication machinery at the cell nucleoid.

FamRCA-RACE: A ROLLING CIRCLE AMPLIFICATION RACE FOR ISOLATING A FAMILY OF HOMOLOGOUS cDNAs IN ONE REACTION AND ITS APPLICATION TO OBTAIN NAC GENES TRANSCRIPTION FACTORS FROM CROCUS (CROCUS SATIVUS) FLOWER

We describe an improvement of the RCA-RACE (rolling circle amplification—rapid amplification of cDNA ends) method, called family RCA-RACE (famRCA-RACE). The method is based on the generation of circular cDNA fragments, followed by rolling circle amplification of the circular cDNA using ϕ29 DNA polymerase and the application of PCR using degenerate outworking primers, designed for a conserved region of homologous genes, that allows the isolation of homologous cDNA sequences expressed in the mRNA preparation in a single polymerase chain reaction (PCR). As an example we present the isolation of seven NAC-like transcription factors cDNA sequences expressed in Crocus sativus flower, used for saffron production. Sequence alignment revealed that CsatNAC proteins contain the typical domain structure of plant NAC proteins, consisting of the conserved N-terminal NAC domain used to design the primers and the five subdomains. Phylogenetic analysis revealed that CsatNAC proteins fall in subgroup I of the NAC family of proteins.

Molecular Analysis of Vector Genome Structures After Liver Transduction by Conventional and Self-Complementary Adeno-Associated Viral Serotype Vectors in Murine and Nonhuman Primate Models

Vectors based on several new adeno-associated viral (AAV) serotypes demonstrated strong hepatocyte tropism and transduction efficiency in both small- and large-animal models for liver-directed gene transfer. Efficiency of liver transduction by AAV vectors can be further improved in both murine and nonhuman primate (NHP) animals when the vector genomes are packaged in a self-complementary (sc) format. In an attempt to understand potential molecular mechanism(s) responsible for enhanced transduction efficiency of the sc vector in liver, we performed extensive molecular studies of genome structures of conventional single-stranded (ss) and sc AAV vectors from liver after AAV gene transfer in both mice and NHPs. These included treatment with exonucleases with specific substrate preferences, single-cutter restriction enzyme digestion and polarity-specific hybridization-based vector genome mapping, and bacteriophage phi29 DNA polymerase-mediated and double-stranded circular template-specific rescue of persisted circular genomes. In mouse liver, vector genomes of both genome formats seemed to persist primarily as episomal circular forms, but sc vectors converted into circular forms more rapidly and efficiently. However, the overall differences in vector genome abundance and structure in the liver between ss and sc vectors could not account for the remarkable differences in transduction. Molecular structures of persistent genomes of both ss and sc vectors were significantly more heterogeneous in macaque liver, with noticeable structural rearrangements that warrant further characterizations.

Molecular characterization of a distinct begomovirus species from Vernonia cinerea and its associated DNA-β using the bacteriophage Φ29 DNA polymerase

Vernonia cinerea plants with yellow vein symptoms were collected around crop fields in Madurai. A portion (550 bp) of the AV1 gene amplified using degenerate primers from the total DNA purified from diseased leaf sample was cloned and sequenced. Specific primers derived from the above sequence were used to amplify 2,745 nucleotides with the typical genome organization of begomoviral DNA A (EMBL Accession No. AM182232). Sequence comparison with other begomoviruses revealed the greatest identity (82.4%) with Emilia yellow vein virus (EmYVV-[Fz1]) from China and less than 80% with all other known begomoviruses. The International Committee on Taxonomy of Viruses (ICTV) has therefore recognized Vernonia yellow vein virus (VeYVV) as a distinct begomovirus species. Conventional PCR could not amplify the DNA B or DNA beta from the diseased tissue. However, the beta DNA (1364 bp) associated with the disease was obtained (Accession No. FN435836) by the rolling circle amplification-restriction fragment length polymorphism method (RCA-RFLP) using Phi 29 DNA polymerase. Sequence analysis shows that DNA beta of VeYVV has the highest identity (56.8%) with DNA beta of Sigesbeckia yellow vein Guangxi betasatellite (SibYVGxB-[CN: Gx111:05]) and 56-53% with DNA beta associated with other begomoviruses. This is the first report of the molecular characterization of VeYVV from V. cinerea in India. The complete molecular characterization, phylogenetic analysis, and putative recombination events in VeYVV are reported.

Modelling of a DNA packaging motor

During the assembly of many viruses, a powerful molecular motor packages the genome into a preassembled capsid. The Bacillus subtilis phage ϕ29 is an excellent model system to investigate the DNA packaging mechanism because of its highly efficient in vitro DNA packaging activity and the development of a single-molecule packaging assay. Here we make use of structural and biochemical experimental data to build a physical model of DNA packaging by the ϕ29 DNA packaging motor. Based on the model, various dynamic behaviours such as the packaging rate, pause frequency and slip frequency under different ATP concentrations, ADP concentrations, external loads as well as capsid fillings are studied by using Monte Carlo simulation. Good agreement is obtained between the simulated and available experimental results. Moreover, we make testable predictions that should guide future experiments related to motor function.

ϕ29 DNA Polymerase Active Site: Role of Residue Val250 as Metal–dNTP Complex Ligand and in Protein-Primed Initiation

DNA polymerases require two acidic residues to coordinate metal ions A and B at their polymerisation active site during catalysis of nucleotide incorporation. Crystallographic resolution of varphi29 DNA polymerase ternary complex showed that metal B coordination also depends on the carbonyl group of Val250 that belongs to the highly conserved Dx(2)SLYP motif of eukaryotic-type (family B) DNA polymerases. In addition, multiple sequence alignments have shown the specific conservation of this residue among the DNA polymerases that use a protein as primer. Thus, to ascertain its role in polymerisation, we have analysed the behaviour of single mutations introduced at the corresponding Val250 of varphi29 DNA polymerase. The differences in nucleotide binding affinity shown by mutants V250A and V250F with respect to the wild-type DNA polymerase agree to a role for Val250 as a metal B-dNTP complex ligand. In addition, mutant V250F was severely affected in varphi29 DNA replication because of a large reduction in the catalytic efficiency of the protein-primed reactions. In the light of the varphi29 DNA polymerase structures, a role for Val250 residue in the maintenance of the proper architecture of the enzyme to perform the protein-primed reactions is also proposed.

Cell-free protein synthesis using multiply-primed rolling circle amplification products

Utilizing in vitro transcription and translation (IVTT) to produce small quantities of proteins is convenient but requires a significant supply of pure template DNA. This can be cumbersome, particularly when the method is used for many different templates in a high-throughput manner. Multiply-primed rolling circle amplification (RCA) with [#x03C6]29 DNA polymerase is a simple way to generate large amounts of DNA; however, the products of this amplification method have interruptions in both strands and branched structures. In this study, we tested whether RCA-generated DNA can serve as the template for in vitro transcription. We found that RCA DNA[#x02013]generated transcripts work in coupled in vitro translation with nearly the same efficiency (per nanogram of DNA) as those obtained from purified plasmid. We propose a convenient, single-tube format for template amplification, transcription, and translation.

Functional Importance of Bacteriophage ϕ29 DNA Polymerase Residue Tyr148 in Primer-terminus Stabilisation at the 3′-5′ Exonuclease Active Site

Recent crystallographic resolution of ϕ29 DNA polymerase complexes with ssDNA at its 3′-5′ exonuclease active site has allowed the identification of residues Pro129 and Tyr148 as putative ssDNA ligands, the latter being conserved in the Kx 2 h motif of proofreading family B DNA polymerases. Single substitution of ϕ29 DNA polymerase residue Tyr148 to Ala rendered an enzyme with a reduced capacity to stabilize the binding of the primer terminus at the 3′-5′ exonuclease active site, not having a direct role in the catalysis of the reaction. Analysis of the 3′-5′ exonuclease on primer/template structures showed a critical role for residue Tyr148 in the proofreading of DNA polymerisation errors. In addition, Tyr148 is not involved in coupling polymerisation to strand displacement in contrast to the catalytic residues responsible for the exonuclease reaction, its role being restricted to stabilisation of the frayed 3′ terminus at the exonuclease active site. Altogether, the results lead us to extend the consensus sequence of the above motif of proofreading family B DNA polymerases into Kx 2h x A . The different solutions adopted by proofreading DNA polymerases to stack the 3′ terminus at the exonuclease site are discussed. In addition, the results obtained with mutants at ϕ29 DNA polymerase residue Pro129 allow us to rule out a functional role as ssDNA ligand for this residue.

Long, Processive Enzymatic DNA Synthesis Using 100% Dye-Labeled Terminal Phosphate-Linked Nucleotides

We demonstrate the efficient synthesis of DNA with complete replacement of the four deoxyribonucleoside triphosphate (dNTP) substrates with nucleotides carrying fluorescent labels. A different, spectrally separable fluorescent dye suitable for single molecule fluorescence detection was conjugated to each of the four dNTPs via linkage to the terminal phosphate. Using these modified nucleotides, DNA synthesis by φ29 DNA polymerase was observed to be processive for products thousands of bases in length, with labeled nucleotide affinities and DNA polymerization rates approaching unmodified dNTP levels. Results presented here show the compatibility of these nucleotides for single-molecule, real-time DNA sequencing applications.

Defining Molecular and Domain Boundaries in the Bacteriophage ϕ29 DNA Packaging Motor

Cryo-electron microscopy (cryo-EM) studies of the bacteriophage phi29 DNA packaging motor have delineated the relative positions and molecular boundaries of the 12-fold symmetric head-tail connector, the 5-fold symmetric prohead RNA (pRNA), the ATPase that provides the energy for packaging, and the procapsid. Reconstructions, assuming 5-fold symmetry, were determined for proheads with 174-base, 120-base, and 71-base pRNA; proheads lacking pRNA; proheads with ATPase bound; and proheads in which the packaging motor was missing the connector. These structures are consistent with pRNA and ATPase forming a pentameric motor component around the unique vertex of proheads. They suggest an assembly pathway for the packaging motor and a mechanism for DNA translocation into empty proheads.

Sequencing of the large dsDNA genome of Oryctes rhinoceros nudivirus using multiple displacement amplification of nanogram amounts of virus DNA

The genomic sequence analysis of many large dsDNA viruses is hampered by the lack of enough sample materials. Here, we report a whole genome amplification of the Oryctes rhinoceros nudivirus (OrNV) isolate Ma07 starting from as few as about 10 ng of purified viral DNA by application of ϕ29 DNA polymerase- and exonuclease-resistant random hexamer-based multiple displacement amplification (MDA) method. About 60 μg of high molecular weight DNA with fragment sizes of up to 25 kbp was amplified. A genomic DNA clone library was generated using the product DNA. After 8-fold sequencing coverage, the 127,615 bp of OrNV whole genome was sequenced successfully. The results demonstrate that the MDA-based whole genome amplification enables rapid access to genomic information from exiguous virus samples.

Improved Multiple Displacement Amplification with φ29 DNA Polymerase for Genotyping of Single Human Cells

The ability to genotype multiple loci of single cells would be of significant benefit to investigations of cellular processes such as oncogenesis, meiosis, fertilization, and embryogenesis. We report a simple two-step, single-tube protocol for whole-genome amplification (WGA) from single human cells using components of the GenomiPhi V2 DNA Amplification kit. For the first time, we demonstrate reliable generation of 4-7 microg amplified DNA from a single human cell within 4 h with a minimum amount of artifactual DNA synthesis. DNA amplified from single cells was genotyped for 13 heterozygous short tandem repeats (STRs) and 7 heterozygous single nucleotide polymorphisms (SNPs), and the genotyping results were compared with purified genomic DNA. Accuracy of genotyping (percent of single-cell amplifications genotyped accurately for any particular STR or SNP) varied from 37% to 100% (with an average of 80%) for STRs and from 89% to 100% (averaging 94%) for SNPs. We suggest that the method described in this report is suitable for WGA from single cells, the product of which can be subsequently used for many applications, such as preimplantation genetic analysis (PGD).