Cloning and sequence analysis of the DNA ligase-encoding gene of rhodothermus marinus, and overproduction, purification and characterization of two thermophilic DNA ligases

Sigridur H. Thorbjarnardóttir, Zophonias O. Jónsson*, Ólafur S. Andrésson, Jakob K. Kristjánsson, Gudmundur Eggertsson, Astridur Palsdottir

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

50 Citations (Scopus)

Abstract

In this paper we describe the cloning and sequence analysis of a gene encoding DNA ligase (Lig; EC 6.5.1.2) from the thermophilic bacterium Rhodothermus marinus (Rm). We also describe the overexpression of the Lig-encoding genes of Rm and the thermophile, Thermus scotoductus (Ts), in Escherichia coli, and the purification and characterization of the overproduced Lig. The Rm lig gene encodes a protein of 712 amino acids (aa) with a calculated molecular mass of 79 487 Da. Comparison with published sequences of bacterial Lig revealed significant homology between the NAD+-utilizing Lig, and alignment of their aa sequences revealed several blocks of conserved residues. Both of the purified Lig exhibit nick-closing activity over a wide range of temperatures. Under our assay conditions the Rm Lig was active at 5-75°C with apparent optimal activity above 55°C. The Ts enzyme showed activity at 15-75°C with optimal activity above 65°C. The half-life of the Lig at 91°C was estimated to be 7 min for the Rm Lig and 26 min for the Ts Lig.

Original languageEnglish
Pages (from-to)1-6
Number of pages6
JournalGene
Volume161
Issue number1
DOIs
Publication statusPublished - 1995

Bibliographical note

Funding Information:
We wish to express our thanks to Arngrimur Thorlacius for assistancew ith atomic absorptionm eas-urementsT. his researchw as supportedb y the Icelandic Research Council and the University of Iceland ResearchF und.

Other keywords

  • complementation
  • DNA replication
  • NAD-dependent DNA ligase
  • recombinant DNA
  • T7 overexpression
  • thermophilic bacterium

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