Pitcher, Robert S., Tonkin, Louise M., Green, Andrew J. and Doherty, Aidan J. (2005) Domain structure of a NHEJ DNA repair ligase from Mycobacterium tuberculosis. Journal of Molecular Biology, 351 (3). pp. 531-44. ISSN 0022-2836Full text not available from this repository.
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.
|Keywords:||Amino Acid Sequence Bacterial Proteins/metabolism Base Sequence Binding Sites DNA Primers DNA Repair Electrophoresis, Polyacrylamide Gel Electrophoretic Mobility Shift Assay Ligases/ chemistry/isolation & purification/metabolism Molecular Sequence Data Mycobacterium tuberculosis/ enzymology Protein Conformation Research Support, Non-U.S. Gov't Sequence Homology, Amino Acid|
|Depositing User:||Aidan Doherty|
|Date Deposited:||27 Nov 2006|
|Last Modified:||30 Nov 2012 16:50|
|Google Scholar:||34 Citations|