DNA-Dependent Conformational Changes in the Ku Heterodimer
- 21 March 2008
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 47 (15), 4359-4368
- https://doi.org/10.1021/bi702284c
Abstract
Ionizing radiation induces DNA double-strand breaks which are repaired by the nonhomologous end joining (NHEJ) pathway. NHEJ is initiated upon Ku binding to the DNA ends and facilitating an interaction with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). This heterotrimeric DNA-PK complex is then active as a serine/threonine protein kinase. The molecular mechanisms involved in DNA-PK activation are unknown. Considering the crucial role of Ku in this process, we therefore determined the influence of DNA binding on the structure of the Ku heterodimer. Chemical modification with NHS-biotin and mass spectrometry were used to identify sites of modification. Biotinylation of free Ku revealed several reactive lysines on Ku70 and Ku80 which were reduced or eliminated upon DNA binding. Interestingly, in the predicted C-terminal SAP domain of Ku70, biotinylation patterns were observed which suggest a structural change in this region of the protein induced by DNA binding. Limited proteolytic digests of free and DNA-bound Ku revealed a series of unique peptides, again, indicative of a change in the accessibility of the Ku70 and Ku80 C-terminal domains. A 10 kDa peptide was also identified which was preferentially generated under non-DNA-bound conditions and mapped to the C-terminus of Ku70. These results indicate a DNA-dependent movement or structural change in the C-terminal domains of Ku70 and Ku80 that may contribute to DNA-PKcs binding and activation. These results represent the first demonstration of DNA-induced changes in Ku structure and provide a framework for analysis of DNA-PKcs and the mechanism of DNA-PK activation.Keywords
This publication has 28 references indexed in Scilit:
- Single-stranded DNA ligation and XLF-stimulated incompatible DNA end ligation by the XRCC4-DNA ligase IV complex: influence of terminal DNA sequenceNucleic Acids Research, 2007
- Solution Structure of the C-Terminal Domain of Ku80 Suggests Important Sites for Protein-Protein InteractionsStructure, 2004
- The 3D Solution Structure of the C-terminal Region of Ku86 (Ku86CTR)Journal of Molecular Biology, 2003
- Identification of specific HIV-1 reverse transcriptase contacts to the viral RNA:tRNA complex by mass spectrometry and a primary amine selective reagentProceedings of the National Academy of Sciences, 2002
- Specific interaction of IP6 with human Ku70/80, the DNA-binding subunit of DNA-PKThe EMBO Journal, 2002
- The Three-dimensional Structure of the C-terminal DNA-binding Domain of Human Ku70Journal of Biological Chemistry, 2001
- Structure of the Ku heterodimer bound to DNA and its implications for double-strand break repairNature, 2001
- Prokaryotic Homologs of the Eukaryotic DNA-End-Binding Protein Ku, Novel Domains in the Ku Protein and Prediction of a Prokaryotic Double-Strand Break Repair SystemGenome Research, 2001
- Ku Recruits the XRCC4-Ligase IV Complex to DNA EndsMolecular and Cellular Biology, 2000
- Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDaAnalytical Biochemistry, 1987