The most important clusters are found between residues 2609 and 2647 (ABCDE cluster) and between residues 2023 and 2056 (PQR cluster). Thr2647 was diminished, while Ser2056 was phosphorylated to normal levels. This resulted in severely reduced levels of Artemis nuclease activity in vivo and in vitro. We therefore conclude that the Ku80 carboxy terminus is important to support DNA-PKCSautophosphorylation at specific sites, which facilitates DNA end processing by the Artemis endonuclease and the subsequent joining reaction. DNA double-strand breaks (DSBs) classify among the most detrimental DNA damages, because they have the ability to cause chromosome breakage and translocations. DSBs are readily caused by common exogenous and endogenous agents, including certain oxygen radicals, products of normal metabolism, and ionizing radiation. Effective genomic maintenance therefore requires the presence of a mechanism to repair DSBs. DSB repair in eukaryotic cells is executed by either homologous recombination or by nonhomologous end joining (NHEJ) (15,30). In vertebrates, DSB repair is not only essential for genomic maintenance, but also for the development of a working immune system. The assembly CDK-IN-2 of immunoglobulin or T-cell receptor genes via V(D)J recombination routinely necessitates the introduction and subsequent NHEJ-mediated repair of DSBs (13). The NHEJ pathway facilitates DSB repair by direct ligation of the two ends of a broken DNA molecule (31,36). This requires the sequential loading of several enzymes on both DNA ends. The first event in NHEJ-mediated repair is the association of a Ku70-Ku80 heterodimer (Ku70/80) with each DNA terminus. The Ku70/80 molecule has a ring-shaped structure, made up by the amino-terminal and central domains of both the Ku70 and the Ku80 polypeptides, which exactly fits a DNA helix in its center (33). The DNA-Ku complex functions as a scaffold to attract the other Rabbit polyclonal to ZNF101 known NHEJ factors to the DSB. One of the enzymes that are recruited to the DNA-Ku scaffold is the DNA-dependent protein kinase catalytic subunit (DNA-PKCS), a 469-kDa serine/threonine kinase. The Ku-DNA-PKCScomplex is commonly referred to as DNA-PK. It has been well established that the DNA-PKCSkinase activity is essential for efficient DSB repair, although the mechanism via which DNA-PKCSexerts its function is a matter of current debate (19,35,36). Several autophosphorylation sites have been mapped in the DNA-PKCSprotein. The most important clusters are found between residues 2609 and 2647 (ABCDE cluster) and between residues 2023 and 2056 (PQR cluster). Phosphorylation of the ABCDE cluster was found to specifically stimulate processing and joining of DNA ends, while PQR phosphorylation reduced the level of DNA end processing (35). These findings prompted a model in which DNA-PKCSfunctions as a gatekeeper molecule that regulates access to the DNA termini CDK-IN-2 by changing its phosphorylation status (35). CDK-IN-2 Therefore, DNA-PKCSautophosphorylation may regulate the next steps in the NHEJ process. These next steps include the processing and joining of DNA ends. Processing enzymes prepare nonligatable DNA termini, primarily blocked ends and incompatible single-strand overhangs, for subsequent ligation by the XRCC4/ligase IV complex. The chemistry of the ligation reaction necessitates the addition of 5 phosphate groups or the removal of 3 phosphate groups by polynucleotide kinase (3). Processing of single-strand overhangs is performed by either filling or resection and therefore requires a polymerase or a nuclease, respectively (16,36). Several enzymes with single-strand filling capability, including polymerase , polymerase , and terminal deoxynucleotidyltransferase, have been suggested to function as processing enzymes during NHEJ (16). In contrast, only one nuclease has been conclusively shown to play a role in NHEJ: the endonuclease Artemis. Artemis was first described as an essential contributor to V(D)J recombination, catalyzing the opening of hairpin structures at coding ends (17,21,24). However, because Artemis deficiency not only causes impairment of V(D)J recombination but also increased sensitivity to DSB-inducing ionizing radiation, it was soon recognized that Artemis may act as a processing enzyme for other types of DNA ends during NHEJ as well. The Artemis protein forms a complex with DNA-PK and carries the endonuclease activity that is necessary for the hairpin opening or overhang processing (14,17). It is likely that the Artemis protein is recruited to the repair complex by interaction with the DNA-Ku-DNA-PKCScomplex. Because the NHEJ core factors.
