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    • br Acknowledgment This work was supported

    2021-02-26


    Acknowledgment This work was supported by the Estonian Science Foundation (Grant ETF8862).
    Introduction In higher eukaryotes ionizing-radiation (IR) induced DNA double-strand breaks (DSB) are primarily repaired by the non-homologous end joining (NHEJ) pathway [1]. Ku, a heterodimeric protein with a unique bridge and pillar structure has a very high affinity for DNA termini and binds to the site of the DSB [2]. The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is then recruited to the site of the break, interacting with both the DNA terminus and the Ku heterodimer. The resulting heterotrimeric complex, termed DNA-PK, is active as a serine/threonine protein kinase and can phosphorylate the downstream substrates. As IR induced DSBs often contain other DNA structural damage including thymine glycols, ring fragmentation, 3′ phosphoglycolates, 5′ hydroxyl groups and abasic sites, processing of DNA termini is often necessary before ligation of the double-strand break by the XRCC4/Ligase IV/XLF complex can occur [1], [3]. A variety of enzymes have been implicated in DNA processing, including but not limited to, FEN-1 [4], polynucleotide kinase (PNK) [5], Werner protein [6], [7], MRN [8], DNA polymerase μ and λ [9], [10], and the nuclease Artemis [11]. The results implicating the involvement of Artemis in the NHEJ pathway are based on in vivo data showing that Artemis null difluprednate mg are more sensitive to IR than wild type counterparts [12]. Artemis has DNA-PK dependent endonuclease activity on DNA hairpin structures, and DNA-PK dependent endonuclease processing of 3′ and 5′ single-strand overhangs, with preferential cleavage at the dsDNA/ssDNA junction [11]. It has been suggested that the stimulation of endonuclease activity of Artemis requires binding and phosphorylation by DNA-PK which causes a conformational change in the C-terminal region of Artemis, resulting in relief of Artemis autoinihibition of the endonuclease active site [13]. Other labs have suggested that autophosphorylation of DNA-PK results in a conformational change in the DNA-bound kinase which in turn alters the conformation of DNA such that it can be easily recognized and cleaved by Artemis [14], [15]. While each model differs slightly in mechanism, both models suggest that Artemis endonuclease activity is DNA-PK and ATP dependent. In addition to DNA-PK-dependent endonuclease activity, Artemis has been suggested to possess an intrinsic 5′–3′ DNA-PK-independent exonuclease activity based on in vitro analysis of partially purified preparations of Artemis [11]. Artemis is a member of the β-CASP family, a new group of the metallo-β-lactamase fold superfamily made up of enzymes acting on nucleic acids [16], [17]. Mutational analysis of conserved residues in the catalytic domain disrupts the endonuclease activity of Artemis, although each of these mutants still possesses robust exonuclease activity [18], [19]. This could be a result of Artemis having two independent catalytic sites, one for each of its proposed nuclease activities. However, this would make Artemis a unique enzyme within its family, as metallo-β-lactamase fold enzymes have been classified as only having one active site that has been shown to be the functional catalytic site for all activities [20]. Interestingly, the exonuclease activity has not to date been shown to have a role in vivo, whereas the endonuclease activity has been demonstrated both in vitro and in vivo[1], [21]. In vitro characterization of the exonuclease activity has largely relied on partially purified Artemis protein produced in exogenous systems. A variety of protein purification protocols have been used to obtain purified Artemis, and all include a tagged form of Artemis and affinity chromatography [3], [11], [14], [22], [23], [24]. Some preparations also include an ionic exchange fractionation step, but all final preparations contain both endonuclease and exonuclease activities. Considering the discrepancy between the existing genetic, biochemical and structural data, we pursued the fractionation of Artemis in a baculovirus expression system to ascertain if the exonuclease and endonuclease activities were biochemically separable. We developed a three-step purification protocol which results in the separation of the exonuclease activity from the intrinsic endonuclease activity of Artemis. Biochemical analyses demonstrate unequivocally that the exonuclease activity associated with Artemis is not intrinsic to the Artemis polypeptide. These results are discussed in the context of in vitro and in vivo processing of DNA termini in the NHEJ pathway.