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J. Biol. Chem., Vol. 281, Issue 19, 13412-13423, May 12, 2006

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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^¶||1, Stewart Shuman, An American Cancer Society Research Professor², and James M. Berger³

From the Department of Molecular and Cellular Biology, University of California, Berkeley, California 94720, Molecular Biology Program, ^¶Immunology Program, and ^||Division of Infectious Diseases, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, New York 10021

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 revealed distinct requirements for the adenylylation and end-sealing reactions catalyzed by LigD. We found 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 double-strand break 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.

Received for publication, December 21, 2005 , and in revised form, January 31, 2006.

The atomic coordinates and structure factors (code 1VS0) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by National Institutes of Health Grants GM63611 (to S. S.), AI064693 (to M. S. G.), GM62410 (to J. M. B.), and GM07739 (to J. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Burroughs-Wellcome Fund Investigator in Pathogenesis of Infectious Disease.

² To whom correspondence may be addressed. E-mail: s-shuman{at}ski.mskcc.org. ³ To whom correspondence may be addressed. E-mail: jmberger{at}berkeley.edu.

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