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 angstrom 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 Delta 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.