Crystal structure and nonhomologous end-joining function of the ligase component of Mycobacterium DNA ligase D.
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 angstroms 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 deltaligD 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.
Department of Molecular and Cellular Biology, University of California, Berkeley, California 94720.
David
D
Akey
Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021.
Alexandra
A
Martins
Immunology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021.
Jideofor
J
Aniukwu
Immunology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021; Division of Infectious Diseases, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, New York 10021.
Michael S
MS
Glickman
Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021. Electronic address: s-shuman@ski.mskcc.org.
Stewart
S
Shuman
Department of Molecular and Cellular Biology, University of California, Berkeley, California 94720. Electronic address: jmberger@berkeley.edu.
James M
JM
Berger
D000595:Amino Acid Sequence; D001483:Base Sequence; D003460:Crystallization; D011088:DNA Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary
D011088:DNA Ligases; C493173:DNA ligase D, Mycobacterium tuberculosis