Cloning and expression of a DNA ligase from the hyperthermophilic archaeon Staphylothermus marinus and properties of the enzyme

Seo, Moo Seok; Kim, Yun Jae; Choi, Jeong Hee; Lee, Mi‐Sun; Kim, Jae Hyun; Lee, Jung-Hyun; Kwon, Suk‐Tae

doi:10.1016/j.jbiotec.2006.09.024

Cited by 26 publications

(24 citation statements)

References 28 publications

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“…There are reports where small amount of EDTA are being used in the ligation reaction of DNA ligases [13], [23], [24] but their effect on ligation activity is not demonstrated. However, inhibition of DNA ligase by employing high concentration of EDTA (2 or more times than the concentration of the Mg 2+ ) is reported for many DNA ligases [25]–[27].…”

Section: Discussionmentioning

confidence: 99%

Molecular Characterization of NAD+-Dependent DNA Ligase from Wolbachia Endosymbiont of Lymphatic Filarial Parasite Brugia malayi

2012

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The lymphatic filarial parasite, Brugia malayi contains Wolbachia endobacteria that are essential for development, viability and fertility of the parasite. Therefore, wolbachial proteins have been currently seen as the potential antifilarial drug targets. NAD+-dependent DNA ligase is characterized as a promising drug target in several organisms due to its crucial, indispensable role in DNA replication, recombination and DNA repair. We report here the cloning, expression and purification of NAD+-dependent DNA ligase of Wolbachia endosymbiont of B. malayi (wBm-LigA) for its molecular characterization. wBm-LigA has all the domains that are present in nearly all the eubacterial NAD+-dependent DNA ligases such as N-terminal adenylation domain, OB fold, helix-hairpin-helix (HhH) and BRCT domain except zinc-binding tetracysteine domain. The purified recombinant protein (683-amino acid) was found to be biochemically active and was present in its native form as revealed by the circular dichroism and fluorescence spectra. The purified recombinant enzyme was able to catalyze intramolecular strand joining on a nicked DNA as well as intermolecular joining of the cohesive ends of BstEII restricted lamda DNA in an in vitro assay. The enzyme was localized in the various life-stages of B. malayi parasites by immunoblotting and high enzyme expression was observed in Wolbachia within B. malayi microfilariae and female adult parasites along the hypodermal chords and in the gravid portion as evident by the confocal microscopy. Ours is the first report on this enzyme of Wolbachia and these findings would assist in validating the antifilarial drug target potential of wBm-LigA in future studies.

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Section: Discussionmentioning

confidence: 99%

Molecular Characterization of NAD+-Dependent DNA Ligase from Wolbachia Endosymbiont of Lymphatic Filarial Parasite Brugia malayi

2012

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“…ADPase activities were reported for thermophilic chaperonins (Pyrococcus furiosus, Pyrococcus horikoshii, Methanococcus jannaschii, and Thermoplasma acidophilum) and a DNA ligase (Aeropyrum pernix K1 and Staphylothermus marinus). These proteins were purified in ways similar to that reported here [6][7][8]. Although the chaperonins exhibited an ADPase activity at 80°C, at which the E. coli AK is inactive, it is possible that the chaperonin protected Each 200 lL fraction was diluted 100-fold before being mixed 1 : 1 with 1.5 mgAEmL )1 ADPase-free NtrC1 Cshort-his6 to measure apparent ADP hydrolysis.…”

Section: Discussionmentioning

confidence: 99%

“…One well‐known exception is apyrase (or NTPDase) of eukaryotic cells [5], which breaks both phosphodiester bonds of a nucleotide, hydrolyzing ATP and ADP to AMP and orthophosphate(s). Also, a novel ADPase activity of ATPases from thermophilic organisms, including four different chaperonins [6] and two DNA ligases [7,8], has been reported. It was hypothesized that using ADP as an energy source instead of ATP in thermophilic organisms may be beneficial because ATP is less stable at high temperatures.…”

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confidence: 99%

ADPase activity of recombinantly expressed thermotolerant ATPases may be caused by copurification of adenylate kinase of Escherichia coli

et al. 2009

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ATPases associated with various cellular activities (AAA+ ATPases) form a large family of chaperone-like proteins that use ATP hydrolysis to remodel numerous macromolecular complexes [1]. The NtrC1 protein of Aquifex aeolicus is one such ATPase, belonging to the subfamily whose members are called bacterial enhancer binding proteins (EBPs). EBPs use ATP hydrolysis to activate transcription by the r54-dependent form of RNA polymerase [2]. Although some AAA+ ATPases can operate by hydrolyzing other NTPs or even dNTP and ddNTPs [3,4], they specifically target the phosphodiester bond between b-phosphates and c-phosphates of the nucleotides. They do not hydrolyze ADP, even though such hydrolysis releases free energy similar to that released by cleavage of the bond to the c-phosphate. To our knowledge, such high specificity for the Except for apyrases, ATPases generally target only the c-phosphate of a nucleotide. Some non-apyrase ATPases from thermophilic microorganisms are reported to hydrolyze ADP as well as ATP, which has been described as a novel property of the ATPases from extreme thermophiles. Here, we describe an apparent ADP hydrolysis by highly purified preparations of the AAA+ ATPase NtrC1 from an extremely thermophilic bacterium, Aquifex aeolicus. This activity is actually a combination of the activities of the ATPase and contaminating adenylate kinase (AK) from Escherichia coli, which is present at 1 ⁄ 10 000 of the level of the ATPase. AK catalyzes conversion of two molecules of ADP into AMP and ATP, the latter being a substrate for the ATPase. We raise concern that the observed thermotolerance of E. coli AK and its copurification with thermostable proteins by commonly used methods may confound studies of enzymes that specifically catalyze hydrolysis of nucleoside diphosphates or triphosphates. For example, contamination with E. coli AK may be responsible for reported ADPase activities of the ATPase chaperonins from Pyrococcus furiosus, Pyrococcus horikoshii, Methanococcus jannaschii and Thermoplasma acidophilum; the ATP ⁄ ADP-dependent DNA ligases from Aeropyrum pernix K1 and Staphylothermus marinus; or the reported ATP-dependent activities of ADP-dependent phosphofructokinase of P. furiosus. Purification methods developed to separate NtrC1 ATPase from AK also revealed two distinct forms of the ATPase. One is tightly bound to ADP or GDP and able to bind to Q but not S ion exchange matrixes. The other is nucleotide-free and binds to both Q and S ion exchange matrixes.Abbreviations AAA+ ATPases, ATPases associated with various cellular activities; AK, adenylate kinase; Ap5A, diadenosine pentaphosphate; EBP, enhancer binding protein; Mg-ADP-BeF x , ATP ground state analog composed of a complex of ADP and magnesium and beryllofluoride ions (x denotes uncertain stoichiometry of fluorine atoms); SAXS, small-angle solution X-ray scattering.

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“…Acidithiobacillus ferrooxidans ATP [165] Aeropyrum pernix ATP ADP [166] Ferroplasma acidarmanus ATP [197] Ferroplasma acidophilum ATP NAD + [165] Methanothermobacterium thermoautotrophicum ATP [198] Picrophilus torridus ATP NAD + [165] Pyrococcus horikoshii ATP [169] Pyrococcus furiosus ATP [170] Staphylothermus marinus ATP ADP [167] Sulfophobococcus zilligii ATP ADP GTP [168] Sulfolobus acidocaldarius ATP [165] Sulfolobus shibatae ATP [199] Thermococcus fumicolans ATP NAD + [163] Thermococcus kodakarensis ATP NAD + [162] Thermococcus sp. ATP NAD + [164] Thermococcus sp.…”

Section: Speciesmentioning

confidence: 99%

Rapid progress of DNA replication studies in Archaea, the third domain of life

Ishino

2012

Sci. China Life Sci.

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Archaea, the third domain of life, are interesting organisms to study from the aspects of molecular and evolutionary biology. Archaeal cells have a unicellular ultrastructure without a nucleus, resembling bacterial cells, but the proteins involved in genetic information processing pathways, including DNA replication, transcription, and translation, share strong similarities with those of Eukaryota. Therefore, archaea provide useful model systems to understand the more complex mechanisms of genetic information processing in eukaryotic cells. Moreover, the hyperthermophilic archaea provide very stable proteins, which are especially useful for the isolation of replisomal multicomplexes, to analyze their structures and functions. This review focuses on the history, current status, and future directions of archaeal DNA replication studies.

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Cloning and expression of a DNA ligase from the hyperthermophilic archaeon Staphylothermus marinus and properties of the enzyme

Cited by 26 publications

References 28 publications

Molecular Characterization of NAD+-Dependent DNA Ligase from Wolbachia Endosymbiont of Lymphatic Filarial Parasite Brugia malayi

Molecular Characterization of NAD+-Dependent DNA Ligase from Wolbachia Endosymbiont of Lymphatic Filarial Parasite Brugia malayi

ADPase activity of recombinantly expressed thermotolerant ATPases may be caused by copurification of adenylate kinase of Escherichia coli

Rapid progress of DNA replication studies in Archaea, the third domain of life

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