The Catalytic Subunit of DNA-Dependent Protein Kinase Regulates Proliferation, Telomere Length, and Genomic Stability in Human Somatic Cells

Abstract

The DNA-dependent protein kinase (DNA-PK) complex is a serine/threonine protein kinase comprised of a 469-kDa catalytic subunit (DNA-PK cs ) and the DNA binding regulatory heterodimeric (Ku70/Ku86) complex Ku. DNA-PK functions in the nonhomologous end-joining pathway for the repair of DNA double-stranded breaks (DSBs) introduced by either exogenous DNA damage or endogenous processes, such as lymphoid V(D)J recombination. Not surprisingly, mutations in Ku70, Ku86, or DNA-PK cs result in animals that are sensitive to agents that cause DSBs and that are also immune deficient. While these phenotypes have been validated in several model systems, an extension of them to humans has been missing due to the lack of patients with mutations in any one of the three DNA-PK subunits. The worldwide lack of patients suggests that during mammalian evolution this complex has become uniquely essential in primates. This hypothesis was substantiated by the demonstration that functional inactivation of either Ku70 or Ku86 in human somatic cell lines is lethal. Here we report on the functional inactivation of DNA-PK cs in human somatic cells. Surprisingly, DNA-PK cs does not appear to be essential, although the cell line lacking this gene has profound proliferation and genomic stability deficits not observed for other mammalian systems.The repair of DNA double-stranded breaks (DSBs) is crucial for cell survival, the maintenance of genomic integrity, and the prevention of tumorigenesis (48). DSBs can be caused by exposure to a variety of exogenous agents, including ionizing radiation (IR) and chemotherapeutic agents (reviewed in references 31 and 47), as well as being generated by endogenous cellular mechanisms such as variable (diversity) joining [V(D)J] and class switch recombination, processes required for the maturation of B and T lymphocytes (reviewed in reference 71). Moreover, the terminus of every linear chromosome presents the cell with a naturally occurring double-stranded DNA (dsDNA) end, i.e., a telomere, and this must be specifically regulated in order to ensure the stable maintenance of the genome (reviewed in reference 19). Because of the importance of DNA repair, immune function, and genomic stability for organismal well-being, eukaryotes have evolved at least two major mechanisms for the repair of DSBs: homologous recombination (HR) (reviewed in references 46 and 77) and nonhomologous end joining (NHEJ) (reviewed in reference 47).In lower eukaryotes, HR, in which large stretches of homology between the donor and the recipient DNAs are required, appears to be the major-and often virtually exclusive-pathway for general DNA DSB repair. In higher eukaryotes, HR also plays very important, if not essential, roles in meiosis, sister chromatid exchange, and the repair of stalled or collapsed DNA replication forks (reviewed in references 46 and 77). Moreover, HR is the mechanism that permits gene targeting, a technology used extensively in research and clinical settings (reviewed in reference 32).Despite the importance of HR in...

Phosphorylation of the Cryptochrome 1 C-terminal Tail Regulates Circadian Period Length

“…It has been proposed that one of the other PIKK family members can substitute for DNA-PK in DNA-PKcs null cells (60). Furthermore, most of the members of this family phosphorylate SQ motifs, and it has previously been reported that knockdown of one of these (SMG-1) can cause up-regulation of another (ATM), resulting in enhanced phosphorylation of the nonsense-mediated decay protein hUPF1 (61).…”

Ku86 represses lethal telomere deletion events in human somatic cells

“…General rAAV vector construction and methodology were carried out as described (20,21). The scheme and diagnostic intermediates for gene targeting are shown in detail in Fig.…”

“…Whole-cell extracts were prepared, and total protein was quantitated by using a Bradford assay. For most experiments, 50 g of total protein was applied to an SDS/PAGE and processed for immunoblot analysis (21). Anti-Ku86 (sc-5280) and anti-TRF2 (sc-9143) antibodies were purchased from Santa Cruz Biotechnology, and anti-PARP-1 (556362) antibody was purchased from BD PharMingen.…”

“…3B). As an additional control, an isogenic DNA-PK cs Ϫ/Ϫ HCT116 cell line (21) that is known to be deficient in C-NHEJ and which accumulates spontaneous DNA damage was also scored. These cells showed detectable ␥-H2AX and 53BP1 foci, but the frequency and especially the quantity were significantly less than the Ku86 null cell line (Fig.…”

Reprint of “The clinical impact of deficiency in DNA non-homologous end-joining”

“…which is not reflected in animal models; Ku is essential for viability in human but not rodent cells and loss of DNA-PKcs, although not essential, has a marked impact on proliferative capacity [77,78]. The available evidence suggested that the essential function may lie in telomere maintenance.…”