A single amino acid substitution in DNA-PKcs explains the novel phenotype of the CHO mutant, XR-C2

296

341

The DNA-dependent protein kinase (DNA-PK) plays an essential role in nonhomologous DNA end joining (NHEJ) by initially recognizing and binding to DNA breaks. We have shown that in vitro, purified DNA-PK undergoes autophosphorylation, resulting in loss of activity and disassembly of the kinase complex. Thus, we have suggested that autophosphorylation of the DNA-PK catalytic subunit (DNA-PKcs) may be critical for subsequent steps in DNA repair. Recently, we defined seven autophosphorylation sites within DNA-PKcs. Six of these are tightly clustered within 38 residues of the 4,127-residue protein. Here, we show that while phosphorylation at any single site within the major cluster is not critical for DNA-PK's function in vivo, mutation of several sites abolishes the ability of DNA-PK to function in NHEJ. This is not due to general defects in DNA-PK activity, as studies of the mutant protein indicate that its kinase activity and ability to form a complex with DNA-bound Ku remain largely unchanged. However, analysis of rare coding joints and ends demonstrates that nucleolytic end processing is dramatically reduced in joints mediated by the mutant DNA-PKcs. We therefore suggest that autophosphorylation within the major cluster mediates a conformational change in the DNA-PK complex that is critical for DNA end processing. However, autophosphorylation at these sites may not be sufficient for kinase disassembly.Although DNA is the genetic blueprint for all living organisms, it is extremely sensitive to various forms of damage, including oxidation, hydrolysis, and methylation. Thus, efficient DNA repair systems are essential for the maintenance of chromosomal integrity. DNA double-strand breaks (DSBs) are perhaps the most lethal form of DNA damage. In eukaryotes, primarily two pathways repair DSBs: homologous recombination and nonhomologous DNA end joining (NHEJ). In higher eukaryotes, NHEJ is thought to be the major pathway that repairs these breaks (6,29,37,38). Because NHEJ also functions in developing lymphocytes to repair the DSBs introduced during antigen receptor gene rearrangement, defects in this pathway result in a block in lymphocyte development and the disease known as severe combined immunodeficiency (SCID) (reviewed in references 14 and 29).In the past decade, an intensive research effort has focused on NHEJ, resulting in a reasonable understanding of how DSBs are resolved. There are six known factors which unequivocally function in the NHEJ pathway. Three of these comprise the DNA-dependent protein kinase (DNA-PK) (reviewed in references 19, 20, and 25): the two subunits of the DNA end binding heterodimer Ku and the catalytic subunit of DNA-PK (DNA-PKcs) (18). Two other factors, XRCC4 and DNA ligase IV, form a stable DNA ligase complex (15,28,34). The sixth factor, Artemis, was described in 2001 (35); recent data indicate that it may play an important role in DNA end processing during NHEJ (31).DNA-PK plays a central role in NHEJ because it initially recognizes and binds to damaged DNA and then targets other...

Section: Resultssupporting

confidence: 91%

Section: Discussionmentioning

confidence: 99%

Autophosphorylation of the Catalytic Subunit of the DNA-Dependent Protein Kinase Is Required for Efficient End Processing during DNA Double-Strand Break Repair

Ding

Reddy

Wang

et al. 2003

296

341

“…Signal end joining is variably depressed in different DNA-PKcs-deficient cell lines and in different animal models of DNA-PKcs deficiency (16,26,32,41). Using complementation strategies, we (and others) have shown that V3 cells have a significant defect in signal end joining (11,20 (8,11,18,39). Whereas the TϾala mutant supports substantial signal end joining, the TϾasp mutant does not.…”

Section: Vol 27 2007 Phosphorylation Of T3950 Inactivates Dna-pk 1585mentioning

confidence: 80%

The DNA-Dependent Protein Kinase Catalytic Subunit Is Phosphorylated In Vivo on Threonine 3950, a Highly Conserved Amino Acid in the Protein Kinase Domain

Douglas

Chen

Block

et al. 2007

112

156

The protein kinase activity of the DNA-dependent protein kinase (DNA-PK) is required for the repair of DNA double-strand breaks (DSBs) via the process of nonhomologous end joining (NHEJ). However, to date, the only target shown to be functionally relevant for the enzymatic role of DNA-PK in NHEJ is the large catalytic subunit DNA-PKcs itself. In vitro, autophosphorylation of DNA-PKcs induces kinase inactivation and dissociation of DNA-PKcs from the DNA end-binding component Ku70/Ku80. Phosphorylation within the two previously identified clusters of phosphorylation sites does not mediate inactivation of the assembled complex and only partially regulates kinase disassembly, suggesting that additional autophosphorylation sites may be important for DNA-PK function. Here, we show that DNA-PKcs contains a highly conserved amino acid (threonine 3950) in a region similar to the activation loop or t-loop found in the protein kinase domain of members of the typical eukaryotic protein kinase family. We demonstrate that threonine 3950 is an in vitro autophosphorylation site and that this residue, as well as other previously identified sites in the ABCDE cluster, is phosphorylated in vivo in irradiated cells. Moreover, we show that mutation of threonine 3950 to the phosphomimic aspartic acid abrogates V(D)J recombination and leads to radiation sensitivity. Together, these data suggest that threonine 3950 is a functionally important, DNA damage-inducible phosphorylation site and that phosphorylation of this site regulates the activity of DNA-PKcs.

“…It is less clear why attempts to target exon 10 were unsuccessful. Although some of the mouse knockouts involved functional inactivation of N-terminally located exons (23,35), almost all of the naturally occurring spontaneous mutations in DNA-PK cs occur exclusively in the C-terminal half of the protein and generally in the extreme C terminus (9,14,22,63,69,80). This circumstantial evidence suggests that the N-terminal portion of DNA-PK cs may actually encode the essential region of the protein and that perhaps small, generally undetectable amounts of a C-terminally truncated protein may keep the cells/animals alive.…”

Section: Discussionmentioning

confidence: 99%

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

Ruis

Fattah

Hendrickson

2008

110

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...