Crystal structure and nonhomologous end-joining function of the ligase component of Mycobacterium DNA ligase D

David Akey, Alexandra Martins, Jideofor Aniukwu, Michael S. Glickman, Stewart Shuman, James M. Berger

Research output: Contribution to journalArticlepeer-review

Abstract

DNA ligase D (LigD) is a large polyfunctional enzyme involved in nonhomologous end-joining (NHEJ) in mycobacteria. LigD consists of a C-terminal ATP-dependent ligase domain fused to upstream polymerase and phosphoesterase modules. Here we report the 2.4Å crystal structure of the ligase domain of Mycobacterium LigD, captured as the covalent ligase-AMP intermediate with a divalent metal in the active site. A chloride anion on the protein surface coordinated by the ribose 3′-OH and caged by arginine and lysine side chains is a putative mimetic of the 5′-phosphate at a DNA nick. Structure-guided mutational analysis revealed distinct requirements for the adenylylation and end-sealing reactions catalyzed by LigD. We found that amutation of Mycobacterium LigD that ablates only ligase activity results in decreased fidelity of NHEJ in vivo and a strong bias of mutagenic events toward deletions instead of insertions at the sealed DNA ends. This phenotype contrasts with the increased fidelity of double-strand break repair in ΔligD cells or in a strain in which only the polymerase function of LigD is defective. We surmise that the signature error-prone quality of bacterial NHEJ in vivo arises from a dynamic balance between the end-remodeling and end-sealing steps.

Original languageEnglish (US)
Pages (from-to)13412-13423
Number of pages12
JournalJournal of Biological Chemistry
Volume281
Issue number19
DOIs
StatePublished - May 12 2006
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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