Reactivation of heat-inactivated Ku proteins by heat shock cognate protein HSC73 |
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Authors: | Makoto Ihara Kazuko Shichijo Takashi Kudo Kenzo Ohtsuka |
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Affiliation: | 1. Department of Radioisotope Medicine, Atomic Bomb Disease and Hibakusha Medicine Unit, Atomic Bomb Disease Institute, Nagasaki University, Nagasaki, Japan;2. Department of Tumor and Diagnostic Pathology, Atomic Bomb Disease and Hibakusha Medicine Unit, Atomic Bomb Disease Institute, Nagasaki University, Nagasaki, Japan;3. Laboratory of Cell and Stress Biology, College of Bioscience and Biotechnology, Chubu University, Kasugai, Japan |
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Abstract: | Purpose: Mouse double-stranded DNA-dependent protein kinase (DNA-PK) activity is heat sensitive. Recovery of heat-inactivated DNA repair activity is a problem after combination therapy with radiation and heat. We investigated the mechanism of recovery of heat-inactivated DNA-PK activity.Methods: Hybrid cells containing a fragment of human chromosome 8 in scid cells (RD13B2) were used. DNA-PK activity was measured by an in vitro assay. Immunoprecipitation of the nuclear extract was performed with an anti-Ku80 antibody. Proteins co-precipitated with Ku80 were separated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and detected by Western blotting using anti-heat shock protein (HSP)72 and anti-heat shock cognate protein (HSC)73 antibodies. HSC73 was overexpressed with the pcDNA3.1 vector. Short hairpin (sh)RNA was used to downregulate HSC73 and HSP72. Results: The activity of heat-inactivated DNA-PK recovered to about 50% of control during an additional incubation at 37?°C after heat treatment at 44?°C for 15?min in the presence of cycloheximide (which inhibits de novo protein synthesis). Maximal recovery was observed within 3?h of incubation at 37?°C after heat treatment. Constitutively expressed HSC73, which folds newly synthesized proteins, reached maximal levels 3?h after heat treatment using a co-immunoprecipitation assay with the Ku80 protein. Inhibiting HSC73, but not HSP72, expression with shRNA decreased the recovery of DNA-PK activity after heat treatment. Conclusions: These results suggest that de novo protein synthesis is unnecessary for recovery of some heat-inactivated DNA-PK. Rather, it might be reactivated by the molecular chaperone activity of HSC73, but not HSP72. |
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Keywords: | HSC73 Ku70/80 DNA-PK activity heat molecular chaperone |
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