The DNA-dependent protein kinase catalytic subunit (DNA-PKCS) plays an important role during the repair of DNA double-strand breaks (DSBs). It is recruited to DNA ends in the early stages of the nonhomologous end-joining (NHEJ) process, which mediates DSB repair. To study DNA-PKCS recruitment in vivo, we used a laser system to introduce DSBs in a specified region of the cell nucleus. We show that DNA-PKCS accumulates at DSB sites in a Ku80-dependent manner, and that neither the kinase activity nor the phosphorylation status of DNA-PKCS influences its initial accumulation. However, impairment of both of these functions results in deficient DSB repair and the maintained presence of DNA-PKCS at unrepaired DSBs. The use of photobleaching techniques allowed us to determine that the kinase activity and phosphorylation status of DNA-PKCS influence the stability of its binding to DNA ends. We suggest a model in which DNA-PKCS phosphorylation/autophosphorylation facilitates NHEJ by destabilizing the interaction of DNA-PKCS with the DNA ends.

DNA-dependent protein kinase (DNA-PK), consisting of Ku and DNA-PKcs subunits, is the key component of the non-homologous end-joining (NHEJ) pathway of DNA double strand break (DSB) repair. Although the kinase activity of DNA-PKcs is essential for NHEJ, thus far, no in vivo substrate has been conclusively identified except for an autophosphorylation site on DNA-PKcs itself (threonine 2609). Here we report the ionizing radiation (IR)-induced autophosphorylation of DNA-PKcs at a novel site, serine 2056, the phosphorylation of which is required for the repair of DSBs by NHEJ. Interestingly, IR-induced DNA-PKcs autophosphorylation is regulated in a cell cycle-dependent manner with attenuated phosphorylation in the S phase. In contrast, DNA replication-associated DSBs resulted in DNA-PKcs autophosphorylation and localization to DNA damage sites. These results indicate that although IR-induced DNAPKcs phosphorylation is attenuated in the S phase, DNA-PKcs is preferentially activated by the physiologically relevant DNA replication-associated DSBs at the sites of DNA synthesis.Repair of DNA double strand breaks (DSBs) 1 is critical for the maintenance of genome integrity, cell survival, and prevention of tumorigenesis (1, 2). In higher eukaryotes, non-homologous end joining (NHEJ) and homologous recombination (HR) are the two major pathways for DSB repair (3). HR requires the presence of a sister chromatid and is operational in the late S and G 2 phases of the cell cycle because of the availability of an optimally positioned sister chromatid (4). NHEJ, on other hand, does not depend on the presence of homologous DNA sequences and is the predominant pathway for DSB repair in mammalian cells (5). It was proposed that NHEJ is preferentially used in G 1 and early S phases of the cell cycle (6, 7). However, a recent report indicating that NHEJ-deficient cell lines are radiation-sensitive in all phases of the cell cycle suggests that NHEJ is important throughout the cell cycle (8). Clearly, the exact contribution of NHEJ in different phases of the cell cycle needs to be defined further.The NHEJ pathway of DSB repair requires both the DNAdependent protein kinase (DNA-PK) complex and the XRCC4/ DNA ligase IV complex, as well as possible additional accessory factors (5, 9, 10). DNA-PK, the key component of the NHEJ pathway, is composed of the Ku70/80 heterodimer and the catalytic subunit DNA-PKcs (11). Ku binds to DNA ends with very high affinity and is believed to function as the DNAbinding and regulatory subunit that recruits DNA-PKcs to breaks and stimulates its kinase activity (12, 13). DNA-PKcs is a member of the phosphatidylinositol-3-like kinase family that includes ATM (ataxia-telangiectasia mutated) and 15). Although the biochemical properties of DNA-PK have been extensively studied in vitro, it is still not clear how it functions in vivo in the context of NHEJ. Wild type DNA-PKcs, but not a kinase-dead mutant, is able to rescue the radiation sensitivity and DSB repair defect of DNA-PKcs-defective V3 cells demonstrati...

Radiosensitive T -B -severe combined immunodeficiency (RS-SCID) is caused by defects in the nonhomologous end-joining (NHEJ) DNA repair pathway, which results in failure of functional V(D)J recombination. Here we have identified the first human RS-SCID patient to our knowledge with a DNA-PKcs missense mutation (L3062R). The causative mutation did not affect the kinase activity or DNA end-binding capacity of DNA-PKcs itself; rather, the presence of long P-nucleotide stretches in the immunoglobulin coding joints indicated that it caused insufficient Artemis activation, something that is dependent on Artemis interaction with autophosphorylated DNA-PKcs. Moreover, overall end-joining activity was hampered, suggesting that Artemis-independent DNA-PKcs functions were also inhibited. This study demonstrates that the presence of DNA-PKcs kinase activity is not sufficient to rule out a defect in this gene during diagnosis and treatment of RS-SCID patients. Further, the data suggest that residual DNA-PKcs activity is indispensable in humans.Introduction SCID is an inherited primary immunodeficiency. SCID patients present in the first year of life with severe opportunistic infections, chronic diarrhea, and failure to thrive. The total group of SCID patients can be divided in 2 main categories: those with T -B + SCID, who have a T cell signaling defect (70%), and those with T -B -SCID, who have a defect in V(D)J recombination (30%). V(D)J recombination assembles variable (V), diversity (D), and joining (J) gene segments of the Ig and TCR genes during B and T cell differentiation in order to generate a broad repertoire of antigen-specific receptors. V(D)J recombination starts with introduction of DNA breaks at the border of the gene segments and the flanking recombination signal sequences (RSSs) by the RAG1 and RAG2 proteins (1). The resulting blunt signal ends are ligated directly, forming a signal joint. The hairpin sealed coding ends require further processing before coding joint formation can occur. Recognition and repair of the DNA ends occur via the general nonhomologous end-joining (NHEJ) pathway of DNA double-strand break (DSB) repair (2, 3).DSBs induce ATM kinase activity, which phosphorylates histone H2AX (4), followed by binding of 53BP1, MDC1, and a complex of MRE11, RAD50, and NBS1 (MRN complex) (5, 6). These proteins form a microenvironment that holds together the DNA ends over a relatively large distance but still allows some degree of freedom for movement of the DNA ends and access of NHEJ proteins (7).

XRCC4-like factor (XLF)-also known as Cernunnos-has recently been shown to be involved in non-homologous end-joining (NHEJ), which is the main pathway for the repair of DNA double-strand breaks (DSBs) in mammalian cells. XLF is likely to enhance NHEJ by stimulating XRCC4-ligase IV-mediated joining of DSBs. Here, we report mechanistic details of XLF recruitment to DSBs. Live cell imaging combined with laser micro-irradiation showed that XLF is an early responder to DSBs and that Ku is essential for XLF recruitment to DSBs. Biochemical analysis showed that Ku-XLF interaction occurs on DNA and that Ku stimulates XLF binding to DNA. Unexpectedly, XRCC4 is dispensable for XLF recruitment to DSBs, although photobleaching analysis showed that XRCC4 stabilizes the binding of XLF to DSBs. Our observations showed the direct involvement of XLF in the dynamic assembly of the NHEJ machinery and provide mechanistic insights into DSB recognition. Keywords: Cernunnos; Ku; non-homologous end-joining; XLF; XRCC4 EMBO reports (2008) 9, 91-96.

Unrepaired DNA double-strand breaks can lead to apoptosis or tumorigenesis. In mammals double-strand breaks are repaired mainly by nonhomologous end-joining mediated by the DNA-PK complex. The core protein of this complex, DNA-PKcs, is a DNAdependent serine͞threonine kinase that phosphorylates protein targets as well as itself. Although the (auto)phosphorylation activity has been shown to be essential for repair of both random double-strand breaks and induced breaks at the immunoglobulin locus, the corresponding phosphatase has been elusive. In fact, to date, none of the putative phosphatases in DNA double-strand break repair has been identified. Here we show that protein phosphatase 5 interacts with DNA-PKcs and dephosphorylates with surprising specificity at least two functional sites. Cells with either hypo-or hyperphosphorylation of DNA-PKcs at these sites show increased radiation sensitivity.