skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Atomic Structure and Nonhomologous End-Joining Function of the Polymerase Component of Bacterial DNA Ligase D

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- Angstroms 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-joiningmore » in vivo. Our studies underscore a previously uncharacterized role for the primase-like polymerase family in DNA repair.« less

Authors:
; ; ; ; ; ;
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL) National Synchrotron Light Source
Sponsoring Org.:
Doe - Office Of Science
OSTI Identifier:
914365
Report Number(s):
BNL-78933-2007-JA
Journal ID: ISSN 0027-8424; PNASA6; TRN: US200809%%207
DOE Contract Number:  
DE-AC02-98CH10886
Resource Type:
Journal Article
Journal Name:
Proc Natl Acad Sci USA
Additional Journal Information:
Journal Volume: 103; Journal Issue: 6; Journal ID: ISSN 0027-8424
Country of Publication:
United States
Language:
English
Subject:
36 MATERIALS SCIENCE; CRYSTAL STRUCTURE; DNA; DNA REPAIR; FUNCTIONAL ANALYSIS; IN VIVO; LIGASES; POLYMERASES; national synchrotron light source

Citation Formats

Zhu, H, Nandakumar, J, Aniukwu, J, Wang, L, Glickman, M, Lima, C, and Shuman, S. Atomic Structure and Nonhomologous End-Joining Function of the Polymerase Component of Bacterial DNA Ligase D. United States: N. p., 2006. Web. doi:10.1073/pnas.0509083103.
Zhu, H, Nandakumar, J, Aniukwu, J, Wang, L, Glickman, M, Lima, C, & Shuman, S. Atomic Structure and Nonhomologous End-Joining Function of the Polymerase Component of Bacterial DNA Ligase D. United States. doi:10.1073/pnas.0509083103.
Zhu, H, Nandakumar, J, Aniukwu, J, Wang, L, Glickman, M, Lima, C, and Shuman, S. Sun . "Atomic Structure and Nonhomologous End-Joining Function of the Polymerase Component of Bacterial DNA Ligase D". United States. doi:10.1073/pnas.0509083103.
@article{osti_914365,
title = {Atomic Structure and Nonhomologous End-Joining Function of the Polymerase Component of Bacterial DNA Ligase D},
author = {Zhu, H and Nandakumar, J and Aniukwu, J and Wang, L and Glickman, M and Lima, C and Shuman, S},
abstractNote = {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- Angstroms 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.},
doi = {10.1073/pnas.0509083103},
journal = {Proc Natl Acad Sci USA},
issn = {0027-8424},
number = 6,
volume = 103,
place = {United States},
year = {2006},
month = {1}
}