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 approximately 37A 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.
authors.affiliation: Centro de Investigaciones Biológicas (CIB), Consejo Superior de Investigaciones Científicas (CSIC), Ramiro de Maeztu 9, 28040 Madrid, Spain.
authors.forename: María A
authors.initials: MA
authors.lastname: Recuero-Checa
authors.forename: Andrew S
authors.initials: AS
authors.lastname: Doré
authors.forename: Ernesto
authors.initials: E
authors.lastname: Arias-Palomo
authors.forename: Angel
authors.initials: A
authors.lastname: Rivera-Calzada
authors.forename: Sjors H W
authors.initials: SH
authors.lastname: Scheres
authors.forename: Joseph D
authors.initials: JD
authors.lastname: Maman
authors.forename: Laurence H
authors.initials: LH
authors.lastname: Pearl
authors.forename: Oscar
authors.initials: O
authors.lastname: Llorca
mesh_terms: D004247:DNA / Q000378:metabolism*; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure*; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure*; D006801:Humans; D008854:Microscopy, Electron; D008958:Models, Molecular; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary
chemical_list: D004268:DNA-Binding Proteins; C000607893:LIG4 protein, human; C101467:XRCC4 protein, human; D004247:DNA; D011088:DNA Ligases; D000072481:DNA Ligase ATP