The DNA-dependent protein kinase is inactivated by autophosphorylation of the catalytic subunit.
ABSTRACT The DNA-dependent protein kinase (DNA-PK) requires for activity free ends or other discontinuities in the structure of double strand DNA. In vitro, DNA-PK phosphorylates several transcription factors and other DNA-binding proteins and is thought to function in DNA damage recognition or repair and/or transcription. Here we show that in vitro DNA-PK undergoes autophosphorylation of all three protein subunits (DNA-PKcs, Ku p70 and Ku p80) and that phosphorylation correlates with inactivation of the serine/threonine kinase activity of DNA-PK. Significantly, activity is restored by the addition of purified native DNA-PKcs but not Ku, suggesting that inactivation is due to autophosphorylation of DNA-PKcs. Our data also suggest that autophosphorylation results in dissociation of DNA-PKcs from the Ku-DNA complex. We suggest that autophosphorylation is an important mechanism for the regulation of DNA-PK activity.
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ABSTRACT: The repair of DNA double-strand breaks (DSBs) is critical for maintaining genome stability. Although the non-homologous end joining (NHEJ) pathway frequently results in minor changes in DNA sequence at the break site and occasionally the joining of previously unlinked DNA molecules, it is a major contributor to cell survival following exposure of mammalian cells to agents that cause DSBs. This repair mechanism is conserved in lower eukaryotes and in some prokaryotes although the majority of DSBs are repaired by recombinational repair pathways in these organisms. Here we will describe the biochemical properties of NHEJ factors from bacteria, Saccharomyces cerevisiae and mammals, and how physical and functional interactions among these factors co-ordinate the repair of DSBs.DNA Repair 07/2005; 4(6):639-48. DOI:10.1016/j.dnarep.2004.12.005 · 3.36 Impact Factor
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ABSTRACT: Nonhomologous end-joining (NHEJ) is the predominant pathway that repairs DNA double-strand breaks (DSBs) in mammalian cells. The DNA-dependent protein kinase (DNA-PK), consisting of Ku and DNA-PK catalytic subunit (DNA-PKcs), is activated by DNA in vitro and is required for NHEJ. We report that DNA-PKcs is autophosphorylated at Thr2609 in vivo in a Ku-dependent manner in response to ionizing radiation. Phosphorylated DNA-PKcs colocalizes with both gamma-H2AX and 53BP1 after DNA damage. Mutation of Thr2609 to Ala leads to radiation sensitivity and impaired DSB rejoining. These findings establish that Ku-dependent phosphorylation of DNA-PKcs at Thr2609 is required for the repair of DSBs by NHEJ.Genes & Development 10/2002; 16(18):2333-8. DOI:10.1101/gad.1015202 · 12.64 Impact Factor
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ABSTRACT: The repair of DNA double-strand breaks (DSB) is central to the maintenance of genomic integrity. In tumor cells, the ability to repair DSBs predicts response to radiation and many cytotoxic anti-cancer drugs. DSB repair pathways include homologous recombination and non-homologous end joining (NHEJ). NHEJ is a template-independent mechanism, yet many NHEJ repair products carry limited genetic changes, which suggests that NHEJ includes mechanisms to minimize error. Proteins required for mammalian NHEJ include Ku70/80, the DNA-dependent protein kinase (DNA-PKcs), XLF/Cernunnos and the XRCC4:DNA ligase IV complex. NHEJ also utilizes accessory proteins that include DNA polymerases, nucleases, and other end-processing factors. In yeast, mutations of tyrosyl-DNA phosphodiesterase (TDP1) reduced NHEJ fidelity. TDP1 plays an important role in repair of topoisomerase-mediated DNA damage and 3'-blocking DNA lesions, and mutation of the human TDP1 gene results in an inherited human neuropathy termed SCAN1. We found that human TDP1 stimulated DNA binding by XLF and physically interacted with XLF to form TDP1:XLF:DNA complexes. TDP1:XLF interactions preferentially stimulated TDP1 activity on dsDNA as compared to ssDNA. TDP1 also promoted DNA binding by Ku70/80 and stimulated DNA-PK activity. Because Ku70/80 and XLF are the first factors recruited to the DSB at the onset of NHEJ, our data suggest a role for TDP1 during the early stages of mammalian NHEJ. Published by Elsevier B.V.DNA repair 03/2015; 30. DOI:10.1016/j.dnarep.2015.03.003 · 3.36 Impact Factor