Crystal structure and initial characterization of a novel archaeal-like Holliday junction-resolving enzyme from <i>Thermus thermophilus</i> phage Tth15-6

Ahlqvist, Josefin; Linares-Pastén, Javier A.; Håkansson, Maria; Jasilionis, Andrius; Kwiatkowska-Semrau, Karolina; Friðjónsson, Ólafur H.; Kaczorowska, Anna Karina; Dąbrowski, Sławomir; Aevarsson, Arnthór; Hreggviðsson, Guðmundur Ó.; Al-Karadaghi, Salam; Kaczorowski, Tadeusz; Karlsson, Eva Nordberg

doi:10.1107/s2059798321012298

Cited by 6 publications

(5 citation statements)

References 66 publications

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“…54 6 is derived from Tth16-6 phage which is known for infecting Themus thermophiles. 195 Hje (Holliday Junction Endonuclease). Hje resolvase was initially discovered in S. solfataricus cell extracts and belongs to the nuclease superfamily.…”

Section: Holliday Junctionmentioning

confidence: 99%

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Prokaryotic DNA Crossroads: Holliday Junction Formation and Resolution

Nautiyal,

Thakur

2024

ACS Omega

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Cells are continually exposed to a multitude of internal and external stressors, which give rise to various types of DNA damage. To protect the integrity of their genetic material, cells are equipped with a repertoire of repair proteins that engage in various repair mechanisms, facilitated by intricate networks of protein−protein and protein−DNA interactions. Among these networks is the homologous recombination (HR) system, a molecular repair mechanism conserved in all three domains of life. On one hand, HR ensures high-fidelity, template-dependent DNA repair, while on the other hand, it results in the generation of combinatorial genetic variations through allelic exchange. Despite substantial progress in understanding this pathway in bacteria, yeast, and humans, several critical questions remain unanswered, including the molecular processes leading to the exchange of DNA segments, the coordination of protein binding, conformational switching during branch migration, and the resolution of Holliday Junctions (HJs). This Review delves into our current understanding of the HR pathway in bacteria, shedding light on the roles played by various proteins or their complexes at different stages of HR. In the first part of this Review, we provide a brief overview of the end resection processes and the strand-exchange reaction, offering a concise depiction of the mechanisms that culminate in the formation of HJs. In the latter half, we expound upon the alternative methods of branch migration and HJ resolution more comprehensively and holistically, considering the historical research timelines. Finally, when we consolidate our knowledge about HR within the broader context of genome replication and the emergence of resistant species, it becomes evident that the HR pathway is indispensable for the survival of bacteria in diverse ecological niches.

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Section: Holliday Junctionmentioning

confidence: 99%

“…The crystal structure of Hjc_15-6, a novel HJ-resolving enzyme, was determined at 2.54 Å resolution. HJ_15-6 is derived from Tth16-6 phage which is known for infecting Themus thermophiles …”

Section: Resolution Of the Holliday Junctionmentioning

confidence: 99%

Prokaryotic DNA Crossroads: Holliday Junction Formation and Resolution

Nautiyal,

Thakur

2024

ACS Omega

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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%

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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“…Several novel Thermus phage proteins have been characterized in our laboratory within the VIRUS-X project [ 38 ]. They participate in DNA metabolism as Tt72RecA [ 42 ] and novel archaeal-like Holliday junction-resolving enzyme [ 43 ], or through cell lysis as endolysins [ 44 , 45 , 46 , 47 ]. Furthermore, the enzymes from phages are frequently more robust, simpler in structure, and more efficient than those from the native host.…”

Section: Introductionmentioning

confidence: 99%

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

Dorawa

Werbowy

Plotka

et al. 2022

IJMS

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We present a structural and functional analysis of the DNA polymerase of thermophilic Thermus thermophilus MAT72 phage vB_Tt72. The enzyme shows low sequence identity (<30%) to the members of the type-A family of DNA polymerases, except for two yet uncharacterized DNA polymerases of T. thermophilus phages: φYS40 (91%) and φTMA (90%). The Tt72 polA gene does not complement the Escherichia colipolA− mutant in replicating polA-dependent plasmid replicons. It encodes a 703-aa protein with a predicted molecular weight of 80,490 and an isoelectric point of 5.49. The enzyme contains a nucleotidyltransferase domain and a 3′-5′ exonuclease domain that is engaged in proofreading. Recombinant enzyme with His-tag at the N-terminus was overproduced in E. coli, subsequently purified by immobilized metal affinity chromatography, and biochemically characterized. The enzyme exists in solution in monomeric form and shows optimum activity at pH 8.5, 25 mM KCl, and 0.5 mM Mg2+. Site-directed analysis proved that highly-conserved residues D15, E17, D78, D180, and D184 in 3′-5′ exonuclease and D384 and D615 in the nucleotidyltransferase domain are critical for the enzyme’s activity. Despite the source of origin, the Tt72 DNA polymerase has not proven to be highly thermoresistant, with a temperature optimum at 55 °C. Above 60 °C, the rapid loss of function follows with no activity > 75 °C. However, during heat treatment (10 min at 75 °C), trehalose, trimethylamine N-oxide, and betaine protected the enzyme against thermal inactivation. A midpoint of thermal denaturation at Tm = 74.6 °C (ΔHcal = 2.05 × 104 cal mol−1) and circular dichroism spectra > 60 °C indicate the enzyme’s moderate thermal stability.

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Crystal structure and initial characterization of a novel archaeal-like Holliday junction-resolving enzyme from Thermus thermophilus phage Tth15-6

Cited by 6 publications

References 66 publications

Prokaryotic DNA Crossroads: Holliday Junction Formation and Resolution

Prokaryotic DNA Crossroads: Holliday Junction Formation and Resolution

Functional biology and biotechnology of thermophilic viruses

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

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