Archaeal Nucleic Acid Ligases and Their Potential in Biotechnology

Abstract

With their ability to catalyse the formation of phosphodiester linkages, DNA ligases and RNA ligases are essential tools for many protocols in molecular biology and biotechnology. Currently, the nucleic acid ligases from bacteriophage T4 are used extensively in these protocols. In this review, we argue that the nucleic acid ligases from Archaea represent a largely untapped pool of enzymes with diverse and potentially favourable properties for new and emerging biotechnological applications. We summarise the current state of knowledge on archaeal DNA and RNA ligases, which makes apparent the relative scarcity of information on in vitro activities that are of most relevance to biotechnologists (such as the ability to join blunt- or cohesive-ended, double-stranded DNA fragments). We highlight the existing biotechnological applications of archaeal DNA ligases and RNA ligases. Finally, we draw attention to recent experiments in which protein engineering was used to modify the activities of the DNA ligase from Pyrococcus furiosus and the RNA ligase from Methanothermobacter thermautotrophicus, thus demonstrating the potential for further work in this area.

Figure 1

Structures of the DNA ligases from: (a) Sulfolobus solfataricus (PDB ID 2HIX); (b) Thermococcus sp. 1519 (PDB ID 3RR5); (c) Pyrococcus furiosus (PDB ID 2CFM); and (d) Homo sapiens (PDB ID 1X9N). The positions of the DNA binding domain (DBD), adenylation domain (AdD), and oligonucleotide binding domain (OBD) are indicated in the S. solfataricus structure. While the DBD and AdD occupy equivalent positions in all of the structures, the S. solfataricus enzyme shows an open extended conformation of the OBD, the Thermococcus sp. 1519 enzyme shows a 90° anticlockwise rotation of the OBD relative to the S. solfataricus enzyme (indicated by a black arrow) resulting in the intermediate conformation, and the P. furiosus ligase has a 120° rotation exhibiting the closed conformation. Human DNA ligase 1 is shown bound to a nicked DNA substrate (light green). The C-terminal helix of each enzyme is highlighted in purple.

Figure 2

The Pyrococcus abyssi RNA ligase. (a) The homodimeric structure of the enzyme (PDB ID 2VUG). The four domains of each monomer are coloured individually and labelled. (b) Schematic diagram of the domain boundaries, with amino acid numbering shown.