Broad Nucleotide Cofactor Specificity of DNA Ligase from the Hyperthermophilic Crenarchaeon Hyperthermus Butylicus and Its Evolutionary Significance

Overview

Journal Extremophiles

Publisher Springer

Date 2013 Apr 3

PMID 23546841

Citations 2

Authors

Jun-Hwan Kim

Kang-Keun Lee

Younguk Sun

Gang-Jin Seo

Sung Suk Cho

Suk Hyung Kwon

Suk-Tae Kwon

Affiliations

Soon will be listed here.

Abstract

The nucleotide cofactor specificity of the DNA ligase from the hyperthermophilic crenarchaeon Hyperthermus butylicus (Hbu) was studied to investigate the evolutionary relationship of DNA ligases. The Hbu DNA ligase gene was expressed under control of the T7lac promoter of pTARG in Escherichia coli BL21-CodonPlus(DE3)-RIL. The expressed enzyme was purified using the IMPACT™-CN system (intein-mediated purification with an affinity chitin-binding tag) and cation-ion (Arg-tag) chromatography. The optimal temperature for Hbu DNA ligase activity was 75 °C, and the optimal pH was 8.0 in Tris-HCl. The activity was highly dependent on MgCl2 or MnCl2 with maximal activity above 5 mM MgCl2 and 2 mM MnCl2. Notably, Hbu DNA ligase can use ADP and GTP in addition to ATP. The broad nucleotide cofactor specificity of Hbu DNA ligase might exemplify an undifferentiated ancestral stage in the evolution of DNA ligases. This study provides new evidence for possible evolutionary relationships among DNA ligases.

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