Gene cloning, purification, and characterization of recombinant DNA ligases of the thermophilic archaea Pyrococcus abyssi and Methanobacterium thermoautotrophicum

Zakabunin, A. I.; Kamynina, Tatyana P.; Ходырева, С. Н.; Pyshnaya, I. A.; Pyshnyi, D. V.; Khrapov, E. A.; Филипенко, М. Л.

doi:10.1134/s002689331102021x

Cited by 4 publications

(2 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…S. marinus and M. thermoautotrophicum DNA ligases utilize ATP as nucleotide cofactor, and dATP as a weak effector (Sriskanda et al 2000;Kim et al 2013). DNA ligases from T. fumicolans (Rolland et al 2004), T. kodakaraensis KOD1 (Nakatani et al 2000), and P. abyssi (Zakabunin et al 2011), which are phylogenetically closely related to T. Barophilus, and Haloferax volcanii (Zhao et al 2006) can utilize NAD + as a nucleotide cofactor, as well as ATP. Moreover, DNA ligases from S. shibatae and S. marinus are dependent on ATP/ADP to seal nicks (Lai et al 2002;Seo et al 2007).…”

Section: Discussionmentioning

confidence: 99%

Biochemical characterization of a thermostable DNA ligase from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5

Shi

Huang

Gan

et al. 2019

Appl Microbiol Biotechnol

View full text Add to dashboard Cite

DNA ligases are essential enzymes for DNA replication, repair, and recombination processes by catalyzing a nick-joining reaction in double-stranded DNA. The genome of the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 encodes a putative ATP-dependent DNA ligase (Tba ligase). Herein, we characterized the biochemical properties of the recombinant Tba ligase. The enzyme displays an optimal nick-joining activity at 65~70 o C, and retains its DNA ligation activity even after heated at 100 o C for 2 hours, suggesting the enzyme is a thermostable DNA ligase. The enzyme joins DNA over a wide pH spectrum ranging from 5.0-10.0, and its optimal pH is 6.0-9.0. Tba ligase activity is dependent on a divalent metal ion: Mn 2+ , Mg 2+ or Ca 2+ is an optimal ion for the enzyme activity. The enzyme activity is inhibited by NaCl with high concentrations. Remarkably, Tba ligase activity is independent on an additional nucleotide cofactor. However, ATP is required for Tba ligase activity when lowering the enzyme concentration. Mass spectrometric result shows that the residue K250 of Tba ligase is AMPylation, suggesting that the enzyme is bound to AMP. The substitution of K250 of Tba ligase with Ala abolishes the enzyme activity. These observations suggest that the enzyme is an ATP-dependent DNA ligase. In addition, the mismatches at the first position 3′ to the nick suppress Tba ligase activity more than those at the first position 5′ to the nick. The enzyme also discriminates more effectively mismatches at 3′ to the nick than those at 5′ to the nick in ligation cycling reaction, suggesting that the enzyme might have potential application in single nucleotide polymorphism.

show abstract

Section: Discussionmentioning

confidence: 99%

Biochemical characterization of a thermostable DNA ligase from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5

Shi

Huang

Gan

et al. 2019

Appl Microbiol Biotechnol

View full text Add to dashboard Cite

show abstract

“…PCR was carried out using 5 ng pET-LIGP plasmid [11], containing the DNA ligase nucleotide sequence from P. abyssi, 1× Taq polymerase buffer (65 mM Tris-HCl, pH 9.0, 26 mM (NH 4 ) 2 SO 4 , 3 mM MgSO 4 , 0.5 % Tween 20), 0.2 mM dNTPs, 1 U Taq polymerase, and 0.5 U Pfu polymerase. The cycling conditions for PCR were 95°C for 3 min, followed by 25 cycles at 95°C for 5 s, 60°C for 5 s, and 72°C for 30 s. The resultant 1.79-kb DNA fragments were digested with BamHI/KpnI or NdeI/EcoRI, and cloned into the pQE30 or pET23a vectors, respectively.…”

Section: Cloning Of Pabdbdmentioning

confidence: 99%

DNA-Binding Domain of DNA Ligase from the Thermophilic Archaeon Pyrococcus abyssi: Improving Long-Range PCR and Neutralization of Heparin’s Inhibitory Effect

Oscorbin

Boyarskikh

Zakabunin

et al. 2015

Appl Biochem Biotechnol

Self Cite

View full text Add to dashboard Cite

The DNA-binding domain of the DNA ligase from Pyrococcus abyssi (PabDBD) was mapped and cloned into two expression vectors. The resulting 6X His-tagged proteins, with a predicted molecular mass of approximately 30 kDa, were overexpressed, purified using Ni-NTA resin, and biochemically characterized. Both PabDBD derivatives bound to double-stranded DNA fragments at the temperature range of 40-70 °C, and both were inactivated via heating at 95 °C for 15 min. Complexes of the PabDBD variants with either double- and single-stranded DNA fragments were less stable than the native DNA ligase of P. abyssi. Inclusion of the C-terminally 6X His-tagged PabDBD in the reaction mixture during long-range polymerase chain reaction (PCR) increased the efficacy of amplification and eliminated the inhibitory effect of heparin.

show abstract

Molecular cloning and characterization of Pcal_0039, an ATP-/NAD+-independent DNA ligase from hyperthermophilic archaeon Pyrobaculum calidifontis

Abbas,

Muhammad,

Shakir

et al. 2023

International Journal of Biological Macromolecules

View full text Add to dashboard Cite

Gene cloning, purification, and characterization of recombinant DNA ligases of the thermophilic archaea Pyrococcus abyssi and Methanobacterium thermoautotrophicum

Cited by 4 publications

References 23 publications

Biochemical characterization of a thermostable DNA ligase from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5

Biochemical characterization of a thermostable DNA ligase from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5

DNA-Binding Domain of DNA Ligase from the Thermophilic Archaeon Pyrococcus abyssi: Improving Long-Range PCR and Neutralization of Heparin’s Inhibitory Effect

Molecular cloning and characterization of Pcal_0039, an ATP-/NAD+-independent DNA ligase from hyperthermophilic archaeon Pyrobaculum calidifontis

Contact Info

Product

Resources

About