TY - JOUR
T1 - Interaction of the Ku heterodimer with the DNA ligase IV/Xrcc4 complex and its regulation by DNA-PK
AU - Costantini, Silvia
AU - Woodbine, Lisa
AU - Andreoli, Lucia
AU - Jeggo, Penny A.
AU - Vindigni, Alessandro
N1 - Funding Information:
The authors thank Dr. Caterina Marchetti for providing the human Xrcc4 constructs and the other members of the Proteomics group for useful discussion and technical help. The assistance of Ms. Maria Elena Lopez for the growth and maintenance of the Sf9, M059J, and M059K cells and Christine Harer is gratefully acknowledged. The work was supported by a grant from the Human Frontier Science Program, by a FIRB grant of MIUR, by grant no. 02.00648.ST97 of CNR, Rome, and by a grant from the European Commission (RADRISC).
PY - 2007/6/1
Y1 - 2007/6/1
N2 - DNA non-homologous end-joining (NHEJ) is a major mechanism for repairing DNA double-stranded (ds) breaks in mammalian cells. Here, we characterize the interaction between two key components of the NHEJ machinery, the Ku heterodimer and the DNA ligase IV/Xrcc4 complex. Our results demonstrate that Ku interacts with DNA ligase IV via its tandem BRCT domain and that this interaction is enhanced in the presence of Xrcc4 and dsDNA. Moreover, residues 644-748 of DNA ligase IV encompassing the first BRCT motif are necessary for binding. We show that Ku needs to be in its heterodimeric form to bind DNA ligase IV and that the C-terminal tail of Ku80, which mediates binding to DNA-PKcs, is dispensable for DNA ligase IV recognition. Although the interaction between Ku and DNA ligase IV/Xrcc4 occurs in the absence of DNA-PKcs, the presence of the catalytic subunit of DNA-PK kinase enhances complex formation. Previous studies have shown that DNA-PK kinase activity causes disassembly of DNA-PKcs from Ku at the DNA end. Here, we show that DNA-PK kinase activity also results in disassembly of the Ku/DNA ligase IV/Xrcc4 complex. Collectively, our findings provide novel information on the protein-protein interactions that regulate NHEJ in cells.
AB - DNA non-homologous end-joining (NHEJ) is a major mechanism for repairing DNA double-stranded (ds) breaks in mammalian cells. Here, we characterize the interaction between two key components of the NHEJ machinery, the Ku heterodimer and the DNA ligase IV/Xrcc4 complex. Our results demonstrate that Ku interacts with DNA ligase IV via its tandem BRCT domain and that this interaction is enhanced in the presence of Xrcc4 and dsDNA. Moreover, residues 644-748 of DNA ligase IV encompassing the first BRCT motif are necessary for binding. We show that Ku needs to be in its heterodimeric form to bind DNA ligase IV and that the C-terminal tail of Ku80, which mediates binding to DNA-PKcs, is dispensable for DNA ligase IV recognition. Although the interaction between Ku and DNA ligase IV/Xrcc4 occurs in the absence of DNA-PKcs, the presence of the catalytic subunit of DNA-PK kinase enhances complex formation. Previous studies have shown that DNA-PK kinase activity causes disassembly of DNA-PKcs from Ku at the DNA end. Here, we show that DNA-PK kinase activity also results in disassembly of the Ku/DNA ligase IV/Xrcc4 complex. Collectively, our findings provide novel information on the protein-protein interactions that regulate NHEJ in cells.
KW - BRCA-1 C-terminal (BRCT)
KW - DNA double-strand breaks repair
KW - DNA ligase IV
KW - DNA non-homologous end-joining (NHEJ)
KW - DNA-PK
KW - Ku heterodimer
UR - http://www.scopus.com/inward/record.url?scp=34247602569&partnerID=8YFLogxK
U2 - 10.1016/j.dnarep.2006.12.007
DO - 10.1016/j.dnarep.2006.12.007
M3 - Article
C2 - 17241822
AN - SCOPUS:34247602569
SN - 1568-7864
VL - 6
SP - 712
EP - 722
JO - DNA Repair
JF - DNA Repair
IS - 6
ER -