title: DNA-Binding Domain of DNA Ligase from the Thermophilic Archaeon Pyrococcus abyssi: Improving Long-Range PCR and Neutralization of Heparin's Inhibitory Effect.
abstract: 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.
authors.affiliation: Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russian Federation, osc.igor@gmail.com.
authors.forename: Igor P
authors.initials: IP
authors.lastname: Oscorbin
authors.forename: U A
authors.initials: UA
authors.lastname: Boyarskikh
authors.forename: A I
authors.initials: AI
authors.lastname: Zakabunin
authors.forename: E A
authors.initials: EA
authors.lastname: Khrapov
authors.forename: M L
authors.initials: ML
authors.lastname: Filipenko
mesh_terms: D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D011088:DNA Ligases / Q000737:chemistry* / Q000378:metabolism*; D006493:Heparin / Q000494:pharmacology*; D008958:Models, Molecular; D016133:Polymerase Chain Reaction / Q000379:methods*; D017434:Protein Structure, Tertiary; D043903:Pyrococcus abyssi / Q000201:enzymology*; D019914:Taq Polymerase / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D006493:Heparin; D004247:DNA; D019914:Taq Polymerase; D011088:DNA Ligases