Partial deletions of the autoregulatory C-terminal domain of Artemis and their effect on its nuclease activity.

Artemis is a 692 aa nuclease that is essential for opening hairpins during vertebrate V(D)J recombination. Artemis is also important in the DNA repair of double-strand breaks via the nonhomologous DNA end joining (NHEJ) pathway. Therefore, absence of Artemis has been shown to result not only in the blockage of lymphocyte development in vertebrates, but also sensitivity of organisms and cells to double-strand break-inducing events that arise in the course of normal metabolism. Nonhomologous DNA end joining (NHEJ) is the major pathway for the repair of double-strand DNA breaks in most vertebrate cells during most of the cell cycle, including in resting cells. Artemis is the primary nuclease for resection of damaged DNA at double-strand breaks. Artemis alone is inactive as an endonuclease, though it has 5'-exonuclease activity. The endonuclease activity requires physical interaction with DNA-PKcs and subsequent activation steps. Truncation of the C-terminal half of Artemis permits Artemis to be active, even without DNA-PKcs. Here we create a systematic set of deletions from the Artemis C-terminus to determine the minimal extent of C-terminal deletion for Artemis to function in a DNA-PKcs-independent manner. We discuss these data in the context of recent structural studies. The results will be useful in future studies to determine the full range of functions of the C-terminal region of Artemis in the regulation of its endonuclease activity.

DNA-PKcs chemical inhibition versus genetic mutation: Impact on the junctional repair steps of V(D)J recombination.

Spontaneous DNA-PKcs deficiencies in animals result in a severe combined immunodeficiency (SCID) phenotype because DNA-PKcs is required to activate Artemis for V(D)J recombination coding end hairpin opening. The impact on signal joint formation in these spontaneous mutant mammals is variable. Genetically engineered DNA-PKcs null mice and cells from them show a >1,000-fold reduction in coding joint formation and minimal reduction in signal joint formation during V(D)J recombination. Does chemical inhibition of DNA-PKcs mimic this phenotype? M3814 (also known as Nedisertib) is a potent DNA-PKcs inhibitor. We find here that M3814 causes a quantitative reduction in coding joint formation relative to signal joint formation. The sequences of signal and coding junctions were within normal limits, though rare coding joints showed novel features. The signal junctions generally did not show evidence of resection into the signal ends that is often seen in cells that have genetic defects in DNA-PKcs. Comparison of the chemical inhibition findings here with the known results for spontaneous and engineered DNA-PKcs mutant mammals is informative for considering pharmacologic small molecule inhibition of DNA-PKcs in various types of neoplasia.