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Schematic representation of the bacteriophage Φ29 DNA replication mechanism. Φ29 DNA replication starts non-simultaneously at both DNA ends. The TP/DNA polymerase heterodimer recognizes the p6-complexed replication origins and the DNA polymerase catalyzes the covalent linkage of dAMP to TP residue Ser232 (initiation reaction). After a transition step (not drawn in the figure), the DNA polymerase dissociates from the TP and continues processive elongation coupled to strand displacement. Viral protein p5 binds to the displaced ssDNA strands and is further removed during the polymerization process. Continuous elongation by two DNA polymerases gives rise to the complete duplication of the parental strands. Green ovals: parental TP; black ovals: primer TP; red circles: p6; blue: DNA polymerase; yellow ovals: SSB p5. Linear dsDNA is shown as a double helix. Adapted from de Vega and Salas (2011).
Source publication
Bacillus subtilis phage Φ29 has a linear, double-stranded DNA 19 kb long with an inverted terminal repeat of 6 nucleotides and a protein covalently linked to the 5′ ends of the DNA. This protein, called terminal protein (TP), is the primer for the initiation of replication, a reaction catalyzed by the viral DNA polymerase at the two DNA ends. The D...
Contexts in source publication
Context 1
... of the 29 genome takes place by a process of symmetrical replication in which both origins are used for initiation in a non-simultaneous manner ( Blanco et al., 1989; Figure 1). The protein that acts as primer for the initiation of 29 DNA replication, the so-called TP, is a 266 amino acids protein encoded by the early viral gene 3. ...
Context 2
... 29 TP/DNAP heterodimer recognizes the replication origins at the genome ends (see Figure 1). Such origins are constituted by specific sequences as well as by the parental TP, the major signal in the template for recognition, a fact that suggests that the heterodimer is recruited to the origin through interactions with the parental TP. ...
Context 3
... vitro experiments showed that protein p56 blocks the DNA- binding ability of UDG, and structural data suggest that it does it by mimicking the structure of DNA ( Serrano-Heras et al., 2007;Asensio et al., 2011;Baños-Sanz et al., 2013;Cole et al., 2013). As mentioned above, the mechanism of 29 DNA replication involves the generation of replicative intermediates that contain large stretches of ssDNA (Harding and Ito, 1980;Inciarte et al., 1980; see Figure 1). If uracil residues were present in these stretches of ssDNA by either the misincorporation of deoxyuridine monophosphate (dUMP) during the replication process or by the spontaneous deamination of cytosine in DNA, the elimination of these lesions by the BER pathway would give rise to the loss of terminal viral DNA regions. ...
Similar publications
Many different DNA delivery vehicles have been developed and tested, all with their advantages and disadvantages. The bacteriophage phi29 terminal protein (TP) is covalently linked to the 5’ ends of the phage genome during the DNA replication process. Our approach is to utilize this TP as a platform to incorporate different protein or peptide modul...
Citations
... II) Strand displacement activity, high fidelity, and extreme processivity of Phi29 polymerase allow a highly accurate generation of large fragments during the isothermal reaction. Thus, the product is composed of multiple copies of circular template sequences linked in linear DNA fragment large up to 100 kbp (33). III) The random sequence of six nucleotides can be expected to repeat every 4096 nucleotides (4 6 ) in unknown DNA. ...
Introduction
Colorectal cancer (CRC) is one of the leading oncogenic disorders, both in terms of incidence and mortality. The etiology of the disease is certainly multifactorial. Various risk factors like alcohol consumption, smoking, CRC family history, inflammatory bowel disease, hormone therapy, aspirin/nonsteroidal anti-inflammatory drugs use, higher body mass index, consumption of red and/or processed meat, insufficient physical activity, and decreased intake of fruit and vegetables have been pointed out; however, there is not enough support evidence for a single particular causative mechanism. Recently, gut bacterial microbiota has been shown to influence significantly the pathogenesis of CRC. However, little attention is paid to the putative impact of plasmids in gut flora.
Material and methods
We have designed and tested the workflow for semi-selective isolation and amplification of random circular sequences. The exploitation of rolling circle amplification (RCA) with a random hexamers protocol is crucial for the outcome.
Results
Our results suggest that it is possible to isolate and amplify plasmid DNA from gut flora and further process, sequence, and identify them.
Discussion
Little is known about the interactions between bacterial plasmids and human cells. The collection of plasmid sequencing data and the comparison of CRC patients and healthy control sequences can be the first step to elucidating this phenomenon.
... Phi29 DNAp is a single-subunit DNA polymerase of Bacillus subtilis bacteriophage Phi29 and belongs to B-family polymerases ( Figure 1 A ) ( 20 ) . Phi29 DNAp has high fidelity due to an exonuclease domain for proofreading ( 21 ,22 ) . ...
... Why does a secondary structure interfere with the Phi29 DNAp-mediated replication? Structurally, the strand displacement activity comes from the sharp bend ( ∼90 • ) of the template strand inside the Phi29 DNAp and the steric exclusion of the non-template strand at the narrow channel that is formed by the TPR2, palm and finger domains, together with the exonuclease subdomains ( 20 ) . A single-molecule optical tweezers assay reported that when the sharp template bending was inhibited under tension, the strand displacement was impeded, and Phi29 DNAp underwent a long pause ( 35 ) . ...
R-loops are three-stranded nucleic acid structures that can cause replication stress by blocking replication fork progression. However, the detailed mechanism underlying the collision of DNA replication forks and R-loops remains elusive. To investigate how R-loops induce replication stress, we use single-molecule fluorescence imaging to directly visualize the collision of replicating Phi29 DNA polymerase (Phi29 DNAp), the simplest replication system, and R-loops. We demonstrate that a single R-loop can block replication, and the blockage is more pronounced when an RNA–DNA hybrid is on the non-template strand. We show that this asymmetry results from secondary structure formation on the non-template strand, which impedes the progression of Phi29 DNAp. We also show that G-quadruplex formation on the displaced single-stranded DNA in an R-loop enhances the replication stalling. Moreover, we observe the collision between Phi29 DNAp and RNA transcripts synthesized by T7 RNA polymerase (T7 RNAp). RNA transcripts cause more stalling because of the presence of T7 RNAp. Our work provides insights into how R-loops impede DNA replication at single-molecule resolution.
... Hence, termination of exonuclease activity in phi29 is necessary when using polymer-tagged nucleotides. D12, E14, Y59, H61, N62, D66, F69, K143, Y148, Y165, and D169 have been reported to interact directly with single-stranded DNA (ssDNA), resulting in the formation of an active center for exonuclease activity (Esteban et al., 1994;Eisenbrandt et al., 2002;Wang et al., 2005;Perez-Arnaiz et al., 2009;Salas et al., 2016). D12A and D66A mutations have been shown to eliminate exonuclease activity (Del Prado et al., 2019). ...
In Phi29-α–hemolysin (α-HL) nanopore sequencing systems, a strong electrochemical signal is dependent on a high concentration of salt. However, high salt concentrations adversely affect polymerase activity. Sequencing by synthesis (SBS) requires the use of phi29 polymerase without exonuclease activity to prevent the degradation of modified nucleotide tags; however, the lack of exonuclease activity also affects polymerase processivity. This study aimed to optimize phi29 polymerase for improved salt tolerance and processivity while maintaining its lack of exonuclease activity to meet the requirements of nanopore sequencing. Using salt tolerance compartmentalized self-replication (stCSR) and a microfluidic platform, we obtained 11 mutant sites with enhanced salt tolerance attributes. Sequencing and biochemical analyses revealed that the substitution of conserved amino acids such as G197D, Y369E, T372N, and I378R plays a critical role in maintaining the processivity of exonuclease-deficient phi29 polymerase under high salt conditions. Furthermore, Y369E and T372N have been identified as important determinants of DNA polymerase binding affinity. This study provides insights into optimizing polymerase processability under high-salt conditions for real-time polymerase nanopore sequencing, paving the way for improved performance and applications in nanopore sequencing technologies.
... Phages that can encode their own DNA polymerase are constantly being discovered, and the DNA polymerases of phage phi29, T4, Bam35 and RB69 that have been characterized belong to the family B DNA polymerases [13][14][15][16]. Among all phage-encoded DNA polymerases, phi29 DNA polymerase (phi29 DNAP) is a model replicative DNA polymerase that has been extensively studied at the genetic, physiological and biochemical levels, and has been used in a variety of applications [17,18]. The phi29 DNAP uses a protein as the primer to initiate genome replication, which is a paradigm for protein-primed replication. ...
Background
Identification and characterization of novel, faithful and processive DNA polymerases is a driving force in the development of DNA amplification methods. Purification of proteins from natural phages is often time-consuming, cumbersome and low yielding. Escherichia coli is a host bacterium widely used for the production of recombinant proteins, is the cell factory of choice for in vitro studies of phage protein function.
Results
We expressed the gene encoding Enterococcus faecium phage IME199 DNA polymerase (IME199 DNAP) in Escherichia coli BL21(DE3), and characterized protein function. IME199 DNAP has 3′-5′ exonuclease activity, but does not have 5′-3′ exonuclease activity. In addition, IME199 DNAP has dNTP-dependent 5′-3′ polymerase activity and can amplify DNA at 15–35 °C and a pH range of 5.5–9.5. The amino acid residues Asp30, Glu32, Asp112 and Asp251 are the 3′-5′ exonuclease active sites of IME199 DNAP, while residues Asp596 and Tyr639 are essential for DNA synthesis by IME199 DNAP. More importantly, the IME199 DNAP has strand displacement and processive synthesis capabilities, and can perform rolling circle amplification and multiple displacement amplification with very low error rates (approximately 3.67 × 10–6).
Conclusions
A novel family B DNA polymerase was successfully overproduced in Escherichia coli BL21(DE3). Based on the characterized properties, IME199 DNAP is expected to be developed as a high-fidelity polymerase for DNA amplification at room temperature.
... However, MDA-based WGA results in high DNA yield with higher coverage and amplification fidelity. Isothermal MDA used for WGA is mostly based on Φ29DNAP and derivatives [36,183]. ...
In the same way that specialized DNA polymerases (DNAPs) replicate cellular and viral genomes, only a handful of dedicated proteins from various natural origins as well as engineered versions are appropriate for competent exponential amplification of whole genomes and metagenomes (WGA). Different applications have led to the development of diverse protocols, based on various DNAPs. Isothermal WGA is currently widely used due to the high performance of Φ29 DNA polymerase, but PCR-based methods are also available and can provide competent amplification of certain samples. Replication fidelity and processivity must be considered when selecting a suitable enzyme for WGA. However, other properties, such as thermostability, capacity to couple replication, and double helix unwinding, or the ability to maintain DNA replication opposite to damaged bases, are also very relevant for some applications. In this review, we provide an overview of the different properties of DNAPs widely used in WGA and discuss their limitations and future research directions.
... The DNA replication process in vivo can be divided into initiation, elongation, and termination stages. The initiation of replication of phi29 DNA polymerase in vivo requires terminal protein (TP) as a primer and SSB (single-stranded DNA binding protein) and DBP (double-stranded DNA binding protein) to stabilize the template [20]. However, in RCA techniques in vitro, phi29 DNA polymerase initiates and elongates RCA without the help of other proteins. ...
... In terms of the RCA mechanism in vivo, phi29 DNA polymerase requires the help of TP, SSB, and DBP to guarantee correct initiation. The synthesis of the first five nucleotides is primed by TP as initiation, and the synthesis of the sixth nucleotide will start transition from TP-priming to DNA-priming [20]. During elongation, the displacement and processivity ability can help phi29 DNA polymerase to go through complex structures in templates [25]. ...
Rolling circle amplification is a widely used biosensing technique. Although various secondary structures have been employed in RCA, their effects on RCA efficiency have seldom been reported. Here, we find that stems in circular templates can strongly inhibit RCA, and the primer-stem distance is responsible for the inhibition. Based on the results, we propose an initiation inhibition mechanism and present a design principle for a general RCA assay. Inspired by this mechanism, we further propose a new nucleic acid detection method. The results verify that this method can increase RCA detection sensitivity according to the target recycling principle. Besides DNA detection, it can also achieve single mismatch discrimination of miRNA detection after optimization. This method also shows convenient visualization detection. The initiation inhibition of RCA could be helpful for RCA applications as promising detection techniques.
... In addition, it is based on an isothermal protocol that simplifies its use at the point of care and promotes the development of various applications beyond genomic analysis (15)(16)(17). Most MDA protocols are based on the use of hexamer random primers (RP) and the highly processive DNA polymerase from Bacillus virus Φ 29 (Φ29DNAP) (15,18,19), which can generate very long amplicons, that allow high coverage and detection of single nucleotide polymorphisms (SNP) (20,21). However, MDA also brings some disadvantages, such as the generation of primer-related artifacts, chimeric DNA sequences, or biased poorer amplification of sequences with extreme GC content (22)(23)(24)(25)(26)(27), which may affect uniform genome coverage and sensitivity for the detection of minor alleles or underrepresented sequences (23,28,29). ...
Multiple displacement amplification (MDA) has proven to be a useful technique for obtaining large amounts of DNA from tiny samples in genomics and metagenomics. However, MDA has limitations, such as amplification artifacts and biases that can interfere with subsequent quantitative analysis. To overcome these challenges, alternative methods and engineered DNA polymerase variants have been developed. Here, we present new MDA protocols based on the primer-independent DNA polymerase (piPolB), a replicative-like DNA polymerase endowed with DNA priming and proofreading capacities. These new methods were tested on a mock metagenome containing sequences with high-GC content, followed by deep sequencing. Protocols relying on piPolB as a single enzyme cannot achieve competent amplification due to its limited processivity and the presence of ab initio DNA synthesis. However, an alternative method called piMDA, which combines piPolB with Φ29 DNA polymerases, allows proficient and faithful amplification of the mock metagenome. In addition, the prior denaturation step commonly performed in MDA protocols is dispensable, resulting in a more straightforward protocol. In summary, piMDA outperforms commercial methods in the amplification of metagenomes containing high GC sequences and exhibits similar profiling, error rate, and variant determination as the non-amplified samples.
Graphical abstract
... ORF26, ORF28, ORF29, and, ORF31 were predicted as single-stranded DNA-binding protein (SSB), exonuclease, DNA primase, and DNA helicase, respectively. SSBs bind with high affinity to single-stranded DNA, and DNA exonuclease is a multifunctional hydrolase, which plays a role in DNA replication, DNA mismatch repair, and DNA doublestrand break repair (Keijzers et al., 2016;Salas et al., 2016). DNA primases catalyze the synthesis of short RNA molecules used as primers for DNA polymerases, and are associated with replicative DNA helicases (Frick and Richardson, 2001). ...
Carbapenem-resistant Klebsiella pneumoniae is one of the primary bacterial pathogens that pose a significant threat to global public health because of the lack of available therapeutic options. Phage therapy shows promise as a potential alternative to current antimicrobial chemotherapies. In this study, we isolated a new Siphoviridae phage vB_KpnS_SXFY507 against KPC-producing K. pneumoniae from hospital sewage. It had a short latent period of 20 min and a large burst size of 246 phages/cell. The host range of phage vB_KpnS_SXFY507 was relatively broad. It has a wide range of pH tolerance and high thermal stability. The genome of phage vB_KpnS_SXFY507 was 53,122 bp in length with a G + C content of 49.1%. A total of 81 open-reading frames (ORFs) and no virulence or antibiotic resistance related genes were involved in the phage vB_KpnS_SXFY507 genome. Phage vB_KpnS_SXFY507 showed significant antibacterial activity in vitro . The survival rate of Galleria mellonella larvae inoculated with K. pneumoniae SXFY507 was 20%. The survival rate of K. pneumonia -infected G. mellonella larvae was increased from 20 to 60% within 72 h upon treatment with phage vB_KpnS_SXFY507. In conclusion, these findings indicate that phage vB_KpnS_SXFY507 has the potential to be used as an antimicrobial agent for the control of K. pneumoniae .
... This machinery consists of Φ29 replication proteins, such as terminal protein (TP; p3), DNA polymerase (DNAP; p2), singlestranded DNA-binding protein (SSB; p5), and double-stranded DNAbinding protein (DSB; p6; Fig. 7D and E) [148]. Naturally, the TP/DNAP complex specifically recognizes a specific end sequence of Φ29 replication origins, which are also high-affinity binding sequences for the DSB [149]. Here, DNAP catalyzes the covalent linkage between deoxyadenosine monophosphate (dAMP) of linear DNA and serine on TP liposomes encapsulate the TXTL system along with α-hemolysin genes and IPTG. ...
... [150]. Subsequently, DNAP dissociates from the TP/DNAP complex and proceeds with the elongation process coupled with strand displacement (from the 5 ′ -end to the 3 ′ -end of linear DNA) and leads to DNA replication [149]. SSB and DSB can effectively enhance replication efficiency [57]. ...
Bottom-up approaches in creating artificial cells that can mimic natural cells have significant implications for both basic research and translational application. Among various artificial cell models, liposome is one of the most sophisticated systems. By encapsulating proteins and associated biomolecules, they can functionally reconstitute foundational features of biological cells, such as the ability to divide, communicate, and undergo shape deformation. Yet constructing liposome artificial cells from the genetic level, which is central to generate self-sustained systems remains highly challenging. Indeed, many studies have successfully established the expression of gene-coded proteins inside liposomes. Further, recent endeavors to build a direct integration of gene-expressed proteins for reconstituting molecular functions and phenotypes in liposomes have also significantly increased. Thus, this review presents the development of liposome-based artificial cells to demonstrate the process of gene-expressed proteins and their reconstitution to perform desired molecular and cell-like functions. The molecular and cellular phenotypes discussed here include the self-production of membrane phospholipids, division, shape deformation, self-DNA/RNA replication, fusion, and intercellular communication. Together, this review gives a comprehensive overview of gene-expressing liposomes that can stimulate further research of this technology and achieve artificial cells with superior properties in the future.
... However, for apPol, no processivity factor has been identified to date, raising the possibility that apPol might have an intrinsically high affinity for DNA, allowing the enzyme to perform replicative synthesis without a processivity factor as is seen for the bacteriophage phi29 DNA polymerase. 22 We performed primer extension assays under burst conditions with varying concentrations of substrate S1 to determine apPol's affinity for DNA. During the fast phase of product formation, only the fraction of DNA that preforms the binary complex ( Figure 1B, step 2) gets extended. ...
Plasmodium, the causative agent of malaria, belongs to the phylum Apicomplexa. Most apicomplexans, including Plasmodium, contain an essential nonphotosynthetic plastid called the apicoplast that harbors its own genome that is replicated by a dedicated organellar replisome. This replisome employs a single DNA polymerase (apPol), which is expected to perform both replicative and translesion synthesis. Unlike other replicative polymerases, no processivity factor for apPol has been identified. While preliminary structural and biochemical studies have provided an overall characterization of apPol, the kinetic mechanism of apPol's activity remains unknown. We have used transient state methods to determine the kinetics of replicative and translesion synthesis by apPol and show that apPol has low processivity and efficiency while copying undamaged DNA. Moreover, while apPol can bypass oxidatively damaged lesions, the bypass is error-prone. Taken together, our results raise the following question─how does a polymerase with low processivity, efficiency, and fidelity (for translesion synthesis) faithfully replicate the apicoplast organellar DNA within the hostile environment of the human host? We hypothesize that interactions with putative components of the apicoplast replisome and/or an as-yet-undiscovered processivity factor transform apPol into an efficient and accurate enzyme.
