Domain Structure of a NHEJ DNA Repair Ligase from Mycobacterium tuberculosis

• Published in: Journal of molecular biology 2005-08, Vol.351 (3), p.531-544
• Main Authors: Pitcher, Robert S., Tonkin, Louise M., Green, Andrew J., Doherty, Aidan J.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Bacterial Proteins - metabolism; Base Sequence; Binding Sites; DNA Primers; DNA Repair; Electrophoresis, Polyacrylamide Gel; Electrophoretic Mobility Shift Assay; ligase; Ligases - chemistry; Ligases - isolation & purification; Ligases - metabolism; Molecular Sequence Data; Mycobacterium tuberculosis - enzymology; NHEJ; nuclease; polymerase; Protein Conformation; Sequence Homology, Amino Acid
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2005.06.038

A prokaryotic non-homologous end-joining (NHEJ) system for the repair of DNA double-strand breaks (DSBs), composed of a Ku homodimer (Mt-Ku) and a multidomain multifunctional ATP-dependent DNA ligase (Mt-Lig), has been described recently in Mycobacterium tuberculosis. Mt-Lig exhibits polymerase and nuclease activity in addition to DNA ligation activity. These functions were ascribed to putative polymerase, nuclease and ligase domains that together constitute a monomeric protein. Here, the separate polymerase, nuclease and ligase domains of Mt-Lig were cloned individually, over-expressed and the soluble proteins purified to homogeneity. The polymerase domain demonstrated DNA-dependent RNA primase activity, catalysing the synthesis of unprimed oligoribonucleotides on single-stranded DNA templates. The polymerase domain can also extend DNA in a template-dependent manner. This activity was eliminated when the catalytic aspartate residues were replaced with alanine. The ligase domain catalysed the sealing of nicked double-stranded DNA designed to mimic a DSB, consistent with the role of Mt-Lig in NHEJ. Deletion of the active-site lysine residue prevented the formation of an adenylated ligase complex and consequently thwarted ligation. The nuclease domain did not function independently as a 3′-5′ exonuclease. DNA-binding assays revealed that both the polymerase and ligase domains bind DNA in vitro, the latter with considerably higher affinity. Mt-Ku directly stimulated the polymerase and nuclease activities of Mt-Lig. The polymerase domain bound Mt-Ku in vitro, suggesting it may recruit Mt-Lig to Ku-bound DNA in vivo. Consistent with these data, Mt-Ku stimulated the primer extension activity of the polymerase domain, suggestive of a functional interaction relevant to NHEJ-mediated DSB repair processes.