HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

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

This Article Full Text Full Text (PDF) All Versions of this Article: 281/19/13412    most recent M513550200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Akey, D. Articles by Berger, J. M. Search for Related Content PubMed PubMed Citation Articles by Akey, D. Articles by Berger, J. M. Social Bookmarking                      What's this?

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.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				H. Zhu and S. Shuman Bacterial Nonhomologous End Joining Ligases Preferentially Seal Breaks with a 3'-OH Monoribonucleotide J. Biol. Chem., March 28, 2008; 283(13): 8331 - 8339. [Abstract] [Full Text] [PDF]

				J. Aniukwu, M. S. Glickman, and S. Shuman The pathways and outcomes of mycobacterial NHEJ depend on the structure of the broken DNA ends Genes & Dev., February 15, 2008; 22(4): 512 - 527. [Abstract] [Full Text] [PDF]

				H. Zhu and S. Shuman Characterization of Agrobacterium tumefaciens DNA ligases C and D Nucleic Acids Res., June 28, 2007; 35(11): 3631 - 3645. [Abstract] [Full Text] [PDF]

				A. Raymond and S. Shuman Deinococcus radiodurans RNA ligase exemplifies a novel ligase clade with a distinctive N-terminal module that is important for 5'-PO4 nick sealing and ligase adenylylation but dispensable for phosphodiester formation at an adenylylated nick Nucleic Acids Res., February 16, 2007; 35(3): 839 - 849. [Abstract] [Full Text] [PDF]

				L. Yakovleva and S. Shuman Nucleotide Misincorporation, 3'-Mismatch Extension, and Responses to Abasic Sites and DNA Adducts by the Polymerase Component of Bacterial DNA Ligase D J. Biol. Chem., September 1, 2006; 281(35): 25026 - 25040. [Abstract] [Full Text] [PDF]