Atomic structure and nonhomologous end-joining function of the polymerase component of bacterial DNA ligase D

doi:10.1073/pnas.0509083103

Atomic structure and nonhomologous end-joining function of the polymerase component of bacterial DNA ligase D

Molecular Biology, Structural Biology, and Immunology Programs, Sloan–Kettering Institute, New York, NY 10021

Edited by Charles C. Richardson, Harvard Medical School, Boston, MA, and approved December 9, 2005 (received for review October 17, 2005)

Abstract

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 of Pseudomonas 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 of Mycobacterium LigD that uniquely ablates the polymerase activity results in increased fidelity of blunt-end double-strand break repair in vivo by virtue of eliminating nucleotide insertions at the recombination junctions. Thus, LigD Pol is a direct catalyst of mutagenic nonhomologous end-joining in vivo. Our studies underscore a previously uncharacterized role for the primase-like polymerase family in DNA repair.

Footnotes

*To whom correspondence should be addressed. E-mail: s-shuman{at}ski.mskcc.org
Author contributions: H.Z., J.N., J.A., L.K.W., M.S.G., C.D.L., and S.S. designed research; H.Z., J.N., J.A., L.K.W., and C.D.L. performed research; H.Z., J.N., J.A., L.K.W., M.S.G., C.D.L., and S.S. analyzed data; and S.S. wrote the paper.
Conflict of interest statement: No conflicts declared.
This paper was submitted directly (Track II) to the PNAS office.
Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 2FAO, 2FAQ, and 2FAR).
Abbreviations:

DSB,

double-strand break;

NHEJ,

nonhomologous end-joining;

LigD,

DNA ligase D;

Pol,

polymerase domain;

SeMet,

selenomethionine.