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 double-strand break (DSB) repair by nonhomologous end joining (NHEJ) requires the assembly of several proteins on DNA ends. Although biochemical studies have elucidated several aspects of the NHEJ reaction mechanism, much less is known about NHEJ in living cells, mainly because of the inability to visualize NHEJ repair proteins at DNA damage. Here we provide evidence that a pulsed near IR laser can produce DSBs without any visible alterations in the nucleus, and we show that NHEJ proteins accumulate in the irradiated areas. The levels of DSBs and Ku accumulation diminished in time, showing that this approach allows us to study DNA repair kinetics in vivo. Remarkably, the Ku heterodimers on DNA ends were in dynamic equilibrium with Ku70/80 in solution, showing that NHEJ complex assembly is reversible. Accumulation of XRCC4/ligase IV on DSBs depended on the presence of Ku70/80, but not DNA-PK CS. We detected a direct interaction between Ku70 and XRCC4 that could explain these requirements. Our results suggest that this assembly constitutes the core of the NHEJ reaction and that XRCC4 may serve as a flexible tether between Ku70/80 and ligase IV.DNA repair ͉ DNA-dependent protein kinase ͉ double-strand break repair ͉ fluorescence recovery after photobleaching ͉ live cell imaging

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).

No abstract