Open AccessAtomic structure and nonhomologous end-joining function of the polymerase component of bacterial DNA ligase D
Author(s) -
Hui Zhu,
Jayakrishnan Nandakumar,
Jideofor Aniukwu,
Li Kai Wang,
Michael S. Glickman,
Christopher D. Lima,
Stewart Shuman
Publication year - 2006
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.0509083103
Subject(s) - dna ligase , dna polymerase , biology , polymerase , dna repair , dna polymerase i , dna ligases , primer (cosmetics) , dna clamp , dna polymerase mu , dna , dna replication , primase , microbiology and biotechnology , biochemistry , genetics , chemistry , circular bacterial chromosome , gene , reverse transcriptase , polymerase chain reaction , organic chemistry
DNA ligase D (LigD) is a large polyfunctional protein that participates in a recently discovered pathway of nonhomologous end-joining in bacteria. LigD consists of an ATP-dependent ligase domain fused to a polymerase domain (Pol) and a phosphoesterase module. The Pol activity is remarkable for its dependence on manganese, its ability to perform templated and nontemplated primer extension reactions, and its preference for adding ribonucleotides to blunt DNA ends. Here we report the 1.5-Å crystal structure of the Pol domain ofPseudomonas LigD and its complexes with manganese and ATP/dATP substrates, which reveal a minimized polymerase with a two-metal mechanism and a fold similar to that of archaeal DNA primase. Mutational analysis highlights the functionally relevant atomic contacts in the active site. Although distinct nucleoside conformations and contacts for ATP versus dATP are observed in the cocrystals, the functional analysis suggests that the ATP-binding mode is the productive conformation for dNMP and rNMP incorporation. We find that a mutation ofMycobacterium LigD that uniquely ablates the polymerase activity results in increased fidelity of blunt-end double-strand break repairin vivo by virtue of eliminating nucleotide insertions at the recombination junctions. Thus, LigD Pol is a direct catalyst of mutagenic nonhomologous end-joiningin vivo . Our studies underscore a previously uncharacterized role for the primase-like polymerase family in DNA repair.