TY - JOUR
T1 - Structural characterization of filaments formed by human Xrcc4-Cernunnos/XLF complex involved in nonhomologous DNA end-joining
AU - Ropars, Virginie
AU - Drevet, Pascal
AU - Legrand, Pierre
AU - Baconnais, Sonia
AU - Amram, Jeremy
AU - Faure, Guilhem
AU - Márquez, José A.
AU - Piétrement, Olivier
AU - Guerois, Raphaël
AU - Callebaut, Isabelle
AU - Le Cam, Eric
AU - Revy, Patrick
AU - De Villartay, Jean Pierre
AU - Charbonnier, Jean Baptiste
PY - 2011/8/2
Y1 - 2011/8/2
N2 - Cernunnos/XLF is a core protein of the nonhomologous DNA end-joining (NHEJ) pathway that processes the majority of DNA double-strand breaks in mammals. Cernunnos stimulates the final ligation step catalyzed by the complex between DNA ligase IV and Xrcc4 (X4). Here we present the crystal structure of the X4 1-157- Cernunnos 1-224 complex at 5.5-Å resolution and identify the relative positions of the two factors and their binding sites. The X-ray structure reveals a filament arrangement for X4 1-157 and Cernunnos 1-224 homodimers mediated by repeated interactions through their N-terminal head domains. A filament arrangement of the X4-Cernunnos complex was confirmed by transmission electron microscopy analyses both with truncated and full-length proteins. We further modeled the interface and used structure-based site-directed mutagenesis and calorimetry to characterize the roles of various residues at the X4-Cernunnos interface. We identified four X4 residues (Glu 55, Asp 58, Met 61, and Phe 106) essential for the interaction with Cernunnos. These findings provide new insights into the molecular bases for stimulatory and bridging roles of Cernunnos in the final DNA ligation step.
AB - Cernunnos/XLF is a core protein of the nonhomologous DNA end-joining (NHEJ) pathway that processes the majority of DNA double-strand breaks in mammals. Cernunnos stimulates the final ligation step catalyzed by the complex between DNA ligase IV and Xrcc4 (X4). Here we present the crystal structure of the X4 1-157- Cernunnos 1-224 complex at 5.5-Å resolution and identify the relative positions of the two factors and their binding sites. The X-ray structure reveals a filament arrangement for X4 1-157 and Cernunnos 1-224 homodimers mediated by repeated interactions through their N-terminal head domains. A filament arrangement of the X4-Cernunnos complex was confirmed by transmission electron microscopy analyses both with truncated and full-length proteins. We further modeled the interface and used structure-based site-directed mutagenesis and calorimetry to characterize the roles of various residues at the X4-Cernunnos interface. We identified four X4 residues (Glu 55, Asp 58, Met 61, and Phe 106) essential for the interaction with Cernunnos. These findings provide new insights into the molecular bases for stimulatory and bridging roles of Cernunnos in the final DNA ligation step.
KW - DNA repair
KW - Immunodeficiency
KW - Nonhomologous end-joining
KW - Protein-protein interaction
KW - V(D)J recombination
UR - http://www.scopus.com/inward/record.url?scp=79961219743&partnerID=8YFLogxK
U2 - 10.1073/pnas.1100758108
DO - 10.1073/pnas.1100758108
M3 - Article
C2 - 21768349
AN - SCOPUS:79961219743
SN - 0027-8424
VL - 108
SP - 12663
EP - 12668
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 31
ER -