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Papers In Press, published online ahead of print February 13, 2006
Molecular and Cell Biology, Univ of Califonia, Berkeley, Berkeley, CA 94720
Corresponding Author: jmberger{at}berkeley.edu
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 2.4 Å 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 reveals distinct requirements for the adenylation and end-sealing reactions catalyzed by LigD. We find that a mutation 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 DSB repair in ligD 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.
J. Biol. Chem, 10.1074/jbc.M513550200
Submitted on December 21, 2005
Revised on January 31, 2006
Accepted on February 13, 2006
Crystal structure and nonhomologous end joining function of the ligase component of Mycobacterium DNA ligase D
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