Molecular Characterization of a DNA Polymerase from Thermus thermophilus MAT72 Phage vB_Tt72: A Novel Type-A Family Enzyme with Strong Proofreading Activity

Dorawa, Sebastian; Werbowy, Olesia; Plotka, Magdalena; Kaczorowska, Anna-Karina; Makowska, Joanna; Kozłowski, Łukasz P.; Friðjónsson, Ólafur H.; Hreggviðsson, Guðmundur Ó.; Aevarsson, Arnthór; Kaczorowski, Tadeusz

doi:10.3390/ijms23147945

Cited by 5 publications

(6 citation statements)

References 122 publications

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“…PolA, PolB, PolC reference protein sequences were collected from the NCBI virus database (https://www.ncbi.nlm.nih.gov/labs/virus/vssi/#/) and from the respective publications, in particular, the PolA sequences were from [15,24,25]; PolB sequences were from [7], and PolC sequences were from [26] (https://ftp.ncbi.nih.gov/pub/yutinn/jumping_polymerases_2024/). Open reading frames (ORFs) from the 18,382 Caudoviricetes genomes were searched for polymerases using BLASTP with collected reference PolA, PolB, PolC proteins as queries (e-value threshold of 0.0001).…”

Section: Identi Cation Of Phage Dna Polymerasesmentioning

confidence: 99%

Jumping DNA polymerases in bacteriophages

Yutin,

Tolstoy,

Mutz

et al. 2024

Preprint

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Background Viruses with double-stranded (ds) DNA genomes in the realm Duplodnaviria share a conserved structural gene module but show a broad range of variation in their repertoires of DNA replication proteins. Some of the duplodnaviruses encode (nearly) complete replication systems whereas others lack (almost) all genes required for replication, relying on the host replication machinery. DNA polymerases (DNAPs) comprise the centerpiece of the DNA replication apparatus. The replicative DNAPs are classified into 4 unrelated or distantly related families (A-D), with the protein structures and sequences within each family being, generally, highly conserved. More than half of the duplodnaviruses encode a DNAP of family A, B or C. We showed previously that multiple pairs of closely related viruses in the order Crassvirales encode DNAPs of different families.Methods Groups of phages in which DNAP swapping likely occurred were identified as subtrees of a defined depth in a comprehensive evolutionary tree of tailed bacteriophages that included phages with DNAPs of different families. The DNAP swaps were validated by constrained tree analysis that was performed on phylogenetic tree of large terminase subunits, and the phage genomes encoding swapped DNAPs were aligned using Mauve. The structures of the discovered unusual DNAPs were predicted using AlphaFold2.Results We identified four additional groups of tailed phages in the class Caudoviricetes in which the DNAPs apparently were swapped on multiple occasions, with replacements occurring both between families A and B, or A and C, or between distinct subfamilies within the same family. The DNAP swapping always occurs “in situ”, without changes in the organization of the surrounding genes. In several cases, the DNAP gene is the only region of substantial divergence between closely related phage genomes, whereas in others, the swap apparently involved neighboring genes encoding other proteins involved in phage replication. In addition, we identified two previously undetected, highly divergent groups of family A DNAPs that are encoded in some phage genomes along with the main DNAP implicated in genome replication.Conclusions Replacement of the DNAP gene by one encoding a DNAP of a different family occurred on many independent occasions during the evolution of different families of tailed phages, in some cases, resulting in very closely related phages encoding unrelated DNAPs. DNAP swapping was likely driven by selection for avoidance of host antiphage mechanisms targeting the phage DNAP that remain to be identified, and/or by selection against replicon incompatibility.

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Section: Identi Cation Of Phage Dna Polymerasesmentioning

confidence: 99%

Jumping DNA polymerases in bacteriophages

Yutin,

Tolstoy,

Mutz

et al. 2024

Preprint

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“…Several thermophilic viral alternatives to Taq polymerase have been expressed, purified, and characterized ( Table 1 ), including two originating from bacteriophage infecting the genus Thermus . Many cultivated Thermus phage encode annotated family-A DNA polymerase ( polA ) genes, including vB_Tt72 [ 27 ], ϕYS40 [ 28 ], ϕTMA [ 29 ], G20c [ 30 ], P7426 [ 31 ], P2345 [ 31 ], ϕFA [ 32 ], TSP4 [ 33 ], and Tth15-6 [ 34 ]. Of these, the unclassified myoviruses Thermus phage vB_Tt72, phage ϕYS40, and phage ϕTMA are closely related and likely represent a novel genus in the Caudoviricetes [ 27 ].…”

Section: Dna Polymerasesmentioning

confidence: 99%

“…Many cultivated Thermus phage encode annotated family-A DNA polymerase ( polA ) genes, including vB_Tt72 [ 27 ], ϕYS40 [ 28 ], ϕTMA [ 29 ], G20c [ 30 ], P7426 [ 31 ], P2345 [ 31 ], ϕFA [ 32 ], TSP4 [ 33 ], and Tth15-6 [ 34 ]. Of these, the unclassified myoviruses Thermus phage vB_Tt72, phage ϕYS40, and phage ϕTMA are closely related and likely represent a novel genus in the Caudoviricetes [ 27 ]. They have similar polA genes (>90% amino acid identity) with low sequence similarity to other polymerases [ 27 ].…”

Section: Dna Polymerasesmentioning

confidence: 99%

“…Of these, the unclassified myoviruses Thermus phage vB_Tt72, phage ϕYS40, and phage ϕTMA are closely related and likely represent a novel genus in the Caudoviricetes [ 27 ]. They have similar polA genes (>90% amino acid identity) with low sequence similarity to other polymerases [ 27 ]. Biochemical characterization of the vB_Tt72 DNA polymerase revealed proofreading activity even at low dNTP concentrations (0.4 mM), properties that Taq polymerase lacks completely [ 22 ].…”

Section: Dna Polymerasesmentioning

confidence: 99%

“…Biochemical characterization of the vB_Tt72 DNA polymerase revealed proofreading activity even at low dNTP concentrations (0.4 mM), properties that Taq polymerase lacks completely [ 22 ]. However, the purified vB_Tt72 polymerase lost function rapidly above 60°C, and was not thermostable enough for PCR [ 27 ], limiting potential biotechnology applications.…”

Section: Dna Polymerasesmentioning

confidence: 99%

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Functional biology and biotechnology of thermophilic viruses

Doss

Palmer

Mead³

et al. 2023

Essays in Biochemistry

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Viruses have developed sophisticated biochemical and genetic mechanisms to manipulate and exploit their hosts. Enzymes derived from viruses have been essential research tools since the first days of molecular biology. However, most viral enzymes that have been commercialized are derived from a small number of cultivated viruses, which is remarkable considering the extraordinary diversity and abundance of viruses revealed by metagenomic analysis. Given the explosion of new enzymatic reagents derived from thermophilic prokaryotes over the past 40 years, those obtained from thermophilic viruses should be equally potent tools. This review discusses the still-limited state of the art regarding the functional biology and biotechnology of thermophilic viruses with a focus on DNA polymerases, ligases, endolysins, and coat proteins. Functional analysis of DNA polymerases and primase-polymerases from phages infecting Thermus, Aquificaceae, and Nitratiruptor has revealed new clades of enzymes with strong proofreading and reverse transcriptase capabilities. Thermophilic RNA ligase 1 homologs have been characterized from Rhodothermus and Thermus phages, with both commercialized for circularization of single-stranded templates. Endolysins from phages infecting Thermus, Meiothermus, and Geobacillus have shown high stability and unusually broad lytic activity against Gram-negative and Gram-positive bacteria, making them targets for commercialization as antimicrobials. Coat proteins from thermophilic viruses infecting Sulfolobales and Thermus strains have been characterized, with diverse potential applications as molecular shuttles. To gauge the scale of untapped resources for these proteins, we also document over 20,000 genes encoded by uncultivated viral genomes from high-temperature environments that encode DNA polymerase, ligase, endolysin, or coat protein domains.

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Strategies and procedures to generate chimeric DNA polymerases for improved applications

Yu,

Wang

2024

Appl Microbiol Biotechnol

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Chimeric DNA polymerase with notable performance has been generated for wide applications including DNA amplification and molecular diagnostics. This rational design method aims to improve specific enzymatic characteristics or introduce novel functions by fusing amino acid sequences from different proteins with a single DNA polymerase to create a chimeric DNA polymerase. Several strategies prove to be efficient, including swapping homologous domains between polymerases to combine benefits from different species, incorporating additional domains for exonuclease activity or enhanced binding ability to DNA, and integrating functional protein along with specific protein structural pattern to improve thermal stability and tolerance to inhibitors, as many cases in the past decade shown. The conventional protocol to develop a chimeric DNA polymerase with desired traits involves a Design-Build-Test-Learn (DBTL) cycle. This procedure initiates with the selection of a parent polymerase, followed by the identification of relevant domains and devising a strategy for fusion. After recombinant expression and purification of chimeric polymerase, its performance is evaluated. The outcomes of these evaluations are analyzed for further enhancing and optimizing the functionality of the polymerase. This review, centered on microorganisms, briefly outlines typical instances of chimeric DNA polymerases categorized, and presents a general methodology for their creation. Key points • Chimeric DNA polymerase is generated by rational design method. • Strategies include domain exchange and addition of proteins, domains, and motifs. • Chimeric DNA polymerase exhibits improved enzymatic properties or novel functions.

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Molecular Characterization of a DNA Polymerase from Thermus thermophilus MAT72 Phage vB_Tt72: A Novel Type-A Family Enzyme with Strong Proofreading Activity

Cited by 5 publications

References 122 publications

Jumping DNA polymerases in bacteriophages

Jumping DNA polymerases in bacteriophages

Functional biology and biotechnology of thermophilic viruses

Strategies and procedures to generate chimeric DNA polymerases for improved applications

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