Properties of an NAD+-dependent DNA ligase from the hyperthermophile Thermotoga maritima and its application in PCR amplification of long DNA fragments

The thermostable DNA ligase of Thermotoga maritima (Tma DNA ligase) was expressed in Escherichia coli, and purified by heat treatment followed by metal affinity chromatography. Purified Tma DNA ligase exhibited activity on DNA fragments with cohesive termini, and no activity was detected on blunt-end DNA. The ligase reaction required NAD+, and a divalent cation including Mg2+, Mn2+or Ca2+. The highest activity of Tma DNA ligase occurred at 60 °C and pH 8.0 when Hind III digested plasmid was used as substrate. The purified enzyme had a half-life of over 30 min at 95 °C, and retained over 80% of its activity after holding a pH ranging from 7.2 to 8.8 for 1 h at 80 °C. When the enzyme was employed in PCR cycles, Tma DNA ligase promoted the amplification of long DNA fragments from the genomic DNA of T. maritima.