Electron microscopy of Xrcc4 and the DNA ligase IV-Xrcc4 DNA repair complex

María A. Recuero-Checa, Andrew S. Doré, Ernesto Arias-Palomo, Angel Rivera-Calzada, S. H W Scheres, Joseph D. Maman, Laurence H. Pearl, Oscar Llorca

Research output: Contribution to journalArticle

Abstract

The DNA ligase IV-Xrcc4 complex is responsible for the ligation of broken DNA ends in the non-homologous end-joining (NHEJ) pathway of DNA double strand break repair in mammals. Mutations in DNA ligase IV (Lig4) lead to immunodeficiency and radiosensitivity in humans. Only partial structural information for Lig4 and Xrcc4 is available, while the structure of the full-length proteins and their arrangement within the Lig4-Xrcc4 complex is unknown. The C-terminal domain of Xrcc4, whose structure has not been solved, contains phosphorylation sites for DNA-PKcs and is phylogenetically conserved, indicative of a regulatory role in NHEJ. Here, we have purified full length Xrcc4 and the Lig4-Xrcc4 complex, and analysed their structure by single-particle electron microscopy. The three-dimensional structure of Xrcc4 at a resolution of ∼37 Å reveals that the C-terminus of Xrcc4 forms a dimeric globular domain connected to the N-terminus by a coiled-coil. The N- and C-terminal domains of Xrcc4 locate at opposite ends of an elongated molecule. The electron microscopy images of the Lig4-Xrcc4 complex were examined by two-dimensional image processing and a double-labelling strategy, identifying the site of the C-terminus of Xrcc4 and the catalytic core of Lig4 within the complex. The catalytic domains of Lig4 were found to be in the vicinity of the N-terminus of Xrcc4. We provide a first sight of the structural organization of the Lig4-Xrcc4 complex, which suggests that the BRCT domains could provide the link of the ligase to Xrcc4 while permitting some movements of the catalytic domains of Lig4. This arrangement may facilitate the ligation of diverse configurations of damaged DNA.

Original languageEnglish (US)
Pages (from-to)1380-1389
Number of pages10
JournalDNA Repair
Volume8
Issue number12
DOIs
StatePublished - Dec 3 2009
Externally publishedYes

Fingerprint

DNA Ligases
DNA Repair
Electron microscopy
Catalytic Domain
Electron Microscopy
Repair
Ligation
DNA
DNA End-Joining Repair
Joining
Radiation Tolerance
Ligases
Phosphorylation
Mammals
Labeling
Mutation
Image processing
Molecules
DNA Ligase ATP
Proteins

Keywords

  • DNA ligase IV
  • DNA repair
  • Electron microscopy
  • Lig4
  • NHEJ
  • Xrcc4

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

Recuero-Checa, M. A., Doré, A. S., Arias-Palomo, E., Rivera-Calzada, A., Scheres, S. H. W., Maman, J. D., ... Llorca, O. (2009). Electron microscopy of Xrcc4 and the DNA ligase IV-Xrcc4 DNA repair complex. DNA Repair, 8(12), 1380-1389. https://doi.org/10.1016/j.dnarep.2009.09.007

Electron microscopy of Xrcc4 and the DNA ligase IV-Xrcc4 DNA repair complex. / Recuero-Checa, María A.; Doré, Andrew S.; Arias-Palomo, Ernesto; Rivera-Calzada, Angel; Scheres, S. H W; Maman, Joseph D.; Pearl, Laurence H.; Llorca, Oscar.

In: DNA Repair, Vol. 8, No. 12, 03.12.2009, p. 1380-1389.

Research output: Contribution to journalArticle

Recuero-Checa, MA, Doré, AS, Arias-Palomo, E, Rivera-Calzada, A, Scheres, SHW, Maman, JD, Pearl, LH & Llorca, O 2009, 'Electron microscopy of Xrcc4 and the DNA ligase IV-Xrcc4 DNA repair complex', DNA Repair, vol. 8, no. 12, pp. 1380-1389. https://doi.org/10.1016/j.dnarep.2009.09.007
Recuero-Checa MA, Doré AS, Arias-Palomo E, Rivera-Calzada A, Scheres SHW, Maman JD et al. Electron microscopy of Xrcc4 and the DNA ligase IV-Xrcc4 DNA repair complex. DNA Repair. 2009 Dec 3;8(12):1380-1389. https://doi.org/10.1016/j.dnarep.2009.09.007
Recuero-Checa, María A. ; Doré, Andrew S. ; Arias-Palomo, Ernesto ; Rivera-Calzada, Angel ; Scheres, S. H W ; Maman, Joseph D. ; Pearl, Laurence H. ; Llorca, Oscar. / Electron microscopy of Xrcc4 and the DNA ligase IV-Xrcc4 DNA repair complex. In: DNA Repair. 2009 ; Vol. 8, No. 12. pp. 1380-1389.
@article{4b4c89afa59a4a34bc8b51043f10c5d4,
title = "Electron microscopy of Xrcc4 and the DNA ligase IV-Xrcc4 DNA repair complex",
abstract = "The DNA ligase IV-Xrcc4 complex is responsible for the ligation of broken DNA ends in the non-homologous end-joining (NHEJ) pathway of DNA double strand break repair in mammals. Mutations in DNA ligase IV (Lig4) lead to immunodeficiency and radiosensitivity in humans. Only partial structural information for Lig4 and Xrcc4 is available, while the structure of the full-length proteins and their arrangement within the Lig4-Xrcc4 complex is unknown. The C-terminal domain of Xrcc4, whose structure has not been solved, contains phosphorylation sites for DNA-PKcs and is phylogenetically conserved, indicative of a regulatory role in NHEJ. Here, we have purified full length Xrcc4 and the Lig4-Xrcc4 complex, and analysed their structure by single-particle electron microscopy. The three-dimensional structure of Xrcc4 at a resolution of ∼37 {\AA} reveals that the C-terminus of Xrcc4 forms a dimeric globular domain connected to the N-terminus by a coiled-coil. The N- and C-terminal domains of Xrcc4 locate at opposite ends of an elongated molecule. The electron microscopy images of the Lig4-Xrcc4 complex were examined by two-dimensional image processing and a double-labelling strategy, identifying the site of the C-terminus of Xrcc4 and the catalytic core of Lig4 within the complex. The catalytic domains of Lig4 were found to be in the vicinity of the N-terminus of Xrcc4. We provide a first sight of the structural organization of the Lig4-Xrcc4 complex, which suggests that the BRCT domains could provide the link of the ligase to Xrcc4 while permitting some movements of the catalytic domains of Lig4. This arrangement may facilitate the ligation of diverse configurations of damaged DNA.",
keywords = "DNA ligase IV, DNA repair, Electron microscopy, Lig4, NHEJ, Xrcc4",
author = "Recuero-Checa, {Mar{\'i}a A.} and Dor{\'e}, {Andrew S.} and Ernesto Arias-Palomo and Angel Rivera-Calzada and Scheres, {S. H W} and Maman, {Joseph D.} and Pearl, {Laurence H.} and Oscar Llorca",
year = "2009",
month = "12",
day = "3",
doi = "10.1016/j.dnarep.2009.09.007",
language = "English (US)",
volume = "8",
pages = "1380--1389",
journal = "DNA Repair",
issn = "1568-7864",
publisher = "Elsevier",
number = "12",

}

TY - JOUR

T1 - Electron microscopy of Xrcc4 and the DNA ligase IV-Xrcc4 DNA repair complex

AU - Recuero-Checa, María A.

AU - Doré, Andrew S.

AU - Arias-Palomo, Ernesto

AU - Rivera-Calzada, Angel

AU - Scheres, S. H W

AU - Maman, Joseph D.

AU - Pearl, Laurence H.

AU - Llorca, Oscar

PY - 2009/12/3

Y1 - 2009/12/3

N2 - The DNA ligase IV-Xrcc4 complex is responsible for the ligation of broken DNA ends in the non-homologous end-joining (NHEJ) pathway of DNA double strand break repair in mammals. Mutations in DNA ligase IV (Lig4) lead to immunodeficiency and radiosensitivity in humans. Only partial structural information for Lig4 and Xrcc4 is available, while the structure of the full-length proteins and their arrangement within the Lig4-Xrcc4 complex is unknown. The C-terminal domain of Xrcc4, whose structure has not been solved, contains phosphorylation sites for DNA-PKcs and is phylogenetically conserved, indicative of a regulatory role in NHEJ. Here, we have purified full length Xrcc4 and the Lig4-Xrcc4 complex, and analysed their structure by single-particle electron microscopy. The three-dimensional structure of Xrcc4 at a resolution of ∼37 Å reveals that the C-terminus of Xrcc4 forms a dimeric globular domain connected to the N-terminus by a coiled-coil. The N- and C-terminal domains of Xrcc4 locate at opposite ends of an elongated molecule. The electron microscopy images of the Lig4-Xrcc4 complex were examined by two-dimensional image processing and a double-labelling strategy, identifying the site of the C-terminus of Xrcc4 and the catalytic core of Lig4 within the complex. The catalytic domains of Lig4 were found to be in the vicinity of the N-terminus of Xrcc4. We provide a first sight of the structural organization of the Lig4-Xrcc4 complex, which suggests that the BRCT domains could provide the link of the ligase to Xrcc4 while permitting some movements of the catalytic domains of Lig4. This arrangement may facilitate the ligation of diverse configurations of damaged DNA.

AB - The DNA ligase IV-Xrcc4 complex is responsible for the ligation of broken DNA ends in the non-homologous end-joining (NHEJ) pathway of DNA double strand break repair in mammals. Mutations in DNA ligase IV (Lig4) lead to immunodeficiency and radiosensitivity in humans. Only partial structural information for Lig4 and Xrcc4 is available, while the structure of the full-length proteins and their arrangement within the Lig4-Xrcc4 complex is unknown. The C-terminal domain of Xrcc4, whose structure has not been solved, contains phosphorylation sites for DNA-PKcs and is phylogenetically conserved, indicative of a regulatory role in NHEJ. Here, we have purified full length Xrcc4 and the Lig4-Xrcc4 complex, and analysed their structure by single-particle electron microscopy. The three-dimensional structure of Xrcc4 at a resolution of ∼37 Å reveals that the C-terminus of Xrcc4 forms a dimeric globular domain connected to the N-terminus by a coiled-coil. The N- and C-terminal domains of Xrcc4 locate at opposite ends of an elongated molecule. The electron microscopy images of the Lig4-Xrcc4 complex were examined by two-dimensional image processing and a double-labelling strategy, identifying the site of the C-terminus of Xrcc4 and the catalytic core of Lig4 within the complex. The catalytic domains of Lig4 were found to be in the vicinity of the N-terminus of Xrcc4. We provide a first sight of the structural organization of the Lig4-Xrcc4 complex, which suggests that the BRCT domains could provide the link of the ligase to Xrcc4 while permitting some movements of the catalytic domains of Lig4. This arrangement may facilitate the ligation of diverse configurations of damaged DNA.

KW - DNA ligase IV

KW - DNA repair

KW - Electron microscopy

KW - Lig4

KW - NHEJ

KW - Xrcc4

UR - http://www.scopus.com/inward/record.url?scp=70450224460&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=70450224460&partnerID=8YFLogxK

U2 - 10.1016/j.dnarep.2009.09.007

DO - 10.1016/j.dnarep.2009.09.007

M3 - Article

C2 - 19837014

AN - SCOPUS:70450224460

VL - 8

SP - 1380

EP - 1389

JO - DNA Repair

JF - DNA Repair

SN - 1568-7864

IS - 12

ER -