Abstract
The Ku heterodimer plays an essential role in non-homologous end-joining and other cellular processes including transcription, telomere maintenance and apoptosis. While the function of Ku is regulated through its association with other proteins and nucleic acids, the specific composition of these macromolecular complexes and their dynamic response to endogenous and exogenous cellular stimuli are not well understood. Here we use quantitative proteomics to define the composition of Ku multicomponent complexes and demonstrate that they are dramatically altered in response to UV radiation. Subsequent biochemical assays revealed that the presence of DNA ends leads to the substitution of RNA-binding proteins with DNA and chromatin associated factors to create a macromolecular complex poised for DNA repair. We observed that dynamic remodeling of the Ku complex coincided with exit of Ku and other DNA repair proteins from the nucleolus. Microinjection of sheared DNA into live cells as a mimetic for double strand breaks confirmed these findings in vivo.
Publication types
-
Research Support, N.I.H., Extramural
-
Research Support, Non-U.S. Gov't
MeSH terms
-
Antigens, Nuclear / genetics
-
Antigens, Nuclear / metabolism
-
Blotting, Western
-
Cell Line, Tumor
-
Cell Nucleolus / metabolism
-
DNA / genetics
-
DNA / metabolism*
-
DNA End-Joining Repair*
-
DNA Helicases / genetics
-
DNA Helicases / metabolism*
-
DNA-Binding Proteins / genetics
-
DNA-Binding Proteins / metabolism
-
Fluorescent Antibody Technique
-
Gene Expression Profiling / methods
-
Gene Expression Regulation, Neoplastic / radiation effects
-
HeLa Cells
-
Humans
-
Ku Autoantigen
-
Multiprotein Complexes / genetics
-
Multiprotein Complexes / metabolism
-
Protein Binding / genetics
-
Protein Transport / radiation effects
-
Proteome / classification
-
Proteome / genetics
-
Proteome / metabolism
-
Proteomics / methods*
-
Reverse Transcriptase Polymerase Chain Reaction
-
Ribonucleoproteins / genetics
-
Ribonucleoproteins / metabolism
-
Time Factors
-
Ultraviolet Rays
Substances
-
Antigens, Nuclear
-
DNA-Binding Proteins
-
Multiprotein Complexes
-
Proteome
-
Ribonucleoproteins
-
DNA
-
DNA Helicases
-
XRCC5 protein, human
-
Xrcc6 protein, human
-
Ku Autoantigen