The Ku80 carboxy terminus stimulates joining and artemis-mediated processing of DNA ends
March 2009
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Repair of DNA double-strand breaks (DSBs) is predominantly mediated by nonhomologous end joining (NHEJ) in mammalian cells. NHEJ requires binding of the Ku70-Ku80 heterodimer (Ku70/80) to the DNA ends and subsequent recruitment of the DNA-dependent protein kinase catalytic subunit (DNA-PKCS) and the XRCC4/ligase IV complex. Activation of the DNA-PKCS serine/threonine kinase requires an interaction with Ku70/80 and is essential for NHEJ-mediated DSB repair. In contrast to previous models, we found that the carboxy terminus of Ku80 is not absolutely required for the recruitment and activation of DNA-PKCS at DSBs, although cells that harbored a carboxy-terminal deletion in the Ku80 gene were sensitive to ionizing radiation and showed reduced end-joining capacity. More detailed analysis of this repair defect showed that DNA-PKCS autophosphorylation at Thr2647 was diminished, while Ser2056 was phosphorylated to normal levels. This resulted in severely reduced levels of Artemis nuclease activity in vivo and in vitro. We therefore conclude that the Ku80 carboxy terminus is important to support DNA-PKCS autophosphorylation at specific sites, which facilitates DNA end processing by the Artemis endonuclease and the subsequent joining reaction.
- article
- human
- metabolism
- priority journal
- genetics
- enzyme activity
- nonhuman
- animal
- unclassified drug
- DNA repair
- physiology
- protein expression
- protein function
- animal cell
- molecular genetics
- gene deletion
- double stranded DNA
- protein serine threonine kinase
- protein interaction
- phosphorylation
- immunofluorescence
- peptide fragment
- nuclear protein
- DCLRE1C protein
- DNA binding protein
- DNA protein complex
- DNA dependent protein kinase
- Ku antigen
- Ku80 antigen C terminal peptide (720 732)
- Ku80 protein
- Protein kinase
- Ku80 antigen C-terminal peptide (720-732)
- XRCC4 protein
- animal cell culture
- artemis protein
- autophosphorylation
- carboxy terminal sequence
- cell nucleus antigen
- cell survival
- double stranded DNA break
- endonuclease
- enzyme active site
- homologous recombination
- ionizing radiation
- nonhomologous end joining
- nucleotide binding protein
- polydeoxyribonucleotide synthase
- protein purification
