DNAPKcs At the Heart of the DNA Nonhomologous End Joining Machinery

Along with the high-affinity DNA end-binding protein Ku, DNA-PKcs forms the heterotrimeric holoenzyme termed DNA-dependent protein kinase (DNA-PK) [4,25]. DNA-PK was initially identified biochemically as a relatively abundant nuclear protein whose serine/threonine kinase activity is greatly stimulated by linear double-stranded DNA [4]. Subsequently, a series of radiosensitive mutant cell lines defective in DNA double-strand break repair were found to possess mutations in DNA-PKcs or in one of the two Ku subunits [26]. Thus, DNA-PKcs now has a clearly established role in the NHEJ pathway of DNA double-strand break repair. The immune-deficiency of mice lacking functional DNA-PK is due to a defect in V(D)J recombination—a cut-and-paste genome rearrangement process that occurs in developing B and T lymphocytes to generate the antigen-binding diversity of the immunoglobu-lin and T-cell receptor genes, respectively [27].

Recently, it has became clear that DNA-PK is also physically localized to telomeres, the physical caps at the ends of linear eukaryotic chromosomes, and functions there to help prevent the formation of chromosomal end-to-end fusions [28]. Importantly, the kinase activity of DNA-PKcs is essential for its biological functions [29]. When it becomes assembled at a DNA double-strand break, DNA-PK is believed to recruit and/or phosphorylate additional NHEJ factors, such as DNA ligase IV/XRCC4 and the recently identified Artemis protein, to bring about repair of the lesion (Fig. 3) [30,31]. Another in vivo target for DNA-PK that has been identified recently is interferon regulatory factor-3 (IRF-3), which is phosphorylated by DNA-PK during paramyxovirus infection [32].

Figure 3 Model of NHEJ. Upon induction of a DNA double-strand break, the high-affinity DNA end-binding protein Ku is targeted to the site of damage. Ku end binding then permits the recruitment and activation of DNA-PKcs along with the newly identified Artemis. This complex is then believed to act as a scaffold at the site of damage and, through Artemis activation via DNA-PK phosphorylation, process the DNA ends for subsequent ligation by the DNA-ligase IV/XRCC4 complex. Once ligation is complete the complex is cleared from the site of damage by an unknown mechanism that may involve DNA-PK-mediated phosphorylation.

Figure 3 Model of NHEJ. Upon induction of a DNA double-strand break, the high-affinity DNA end-binding protein Ku is targeted to the site of damage. Ku end binding then permits the recruitment and activation of DNA-PKcs along with the newly identified Artemis. This complex is then believed to act as a scaffold at the site of damage and, through Artemis activation via DNA-PK phosphorylation, process the DNA ends for subsequent ligation by the DNA-ligase IV/XRCC4 complex. Once ligation is complete the complex is cleared from the site of damage by an unknown mechanism that may involve DNA-PK-mediated phosphorylation.

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