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Domain structure of a NHEJ DNA repair ligase from Mycobacterium tuberculosis.
Pitcher, Robert S; Tonkin, Louise M; Green, Andrew J; Doherty, Aidan J.
Afiliação
  • Pitcher RS; Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton BN1 9RQ, UK.
J Mol Biol ; 351(3): 531-44, 2005 Aug 19.
Article em En | MEDLINE | ID: mdl-16023671
ABSTRACT
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.
Assuntos
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Base de dados: MEDLINE Assunto principal: Reparo do DNA / Ligases / Mycobacterium tuberculosis Idioma: En Revista: J Mol Biol Ano de publicação: 2005 Tipo de documento: Article País de afiliação: Reino Unido
Buscar no Google
Base de dados: MEDLINE Assunto principal: Reparo do DNA / Ligases / Mycobacterium tuberculosis Idioma: En Revista: J Mol Biol Ano de publicação: 2005 Tipo de documento: Article País de afiliação: Reino Unido