Abstract
Artemis protein has irreplaceable functions in V(D)J recombination and nonhomologous end joining (NHEJ) as a hairpin and 5' and 3' overhang endonuclease. The kinase activity of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is necessary in activating Artemis as an endonuclease. Here we report that three basal phosphorylation sites and 11 DNA-PKcs phosphorylation sites within the mammalian Artemis are all located in the C-terminal domain. All but one of these phosphorylation sites deviate from the SQ or TQ motif of DNA-PKcs that was predicted previously from in vitro phosphorylation studies. Phosphatase-treated mammalian Artemis and Artemis that is mutated at the three basal phosphorylation sites still retain DNA-PKcs-dependent endonucleolytic activities, indicating that basal phosphorylation is not required for the activation. In vivo studies of Artemis lacking the C-terminal domain have been reported to be sufficient to complement V(D)J recombination in Artemis null cells. Therefore, the C-terminal domain may have a negative regulatory effect on the Artemis endonucleolytic activities, and phosphorylation by DNA-PKcs in the C-terminal domain may relieve this inhibition.
Highlights
V(D)J3 recombination is the somatic DNA recombination that occurs in precursors of lymphocytes
Artemis Can Be Phosphorylated at Multiple Positions in the C-terminal Domain by DNA-PKcs—DNA-PKcs is homologous to phosphatidylinositol 3-kinases, yet in vitro it phosphorylates many protein targets on serine and threonine residues [17, 18]
It has been shown that the DNAPKcs kinase activity is necessary to activate Artemis as a hairpin and 5Ј and 3Ј overhang endonuclease [10], and these activities are very likely to be critical to V(D)J recombination and nonhomologous end joining (NHEJ) in vivo
Summary
Oligonucleotides—The 46-nt hairpin substrate (YM-164) has been described previously (Fig. 4A of Ref. 13). Protein Expression Constructs—Wild-type Artemis expression plasmids were constructed as described [13]. For the ArtemisFLAG baculovirus, full-length Artemis containing a SapI site in front of its stop codon was generated and cloned into pENTR1a-TEV (a modified version pENTR1a (Invitrogen)). The resulting plasmid was digested with SapI and ligated back together to generate an in-frame fusion to the TEV cleavage site and the FLAG tag at the C terminus. This construct was used in the Bac-to-Bac system (Invitrogen) to generate the baculovirus following the manufacturer’s recommendation. DNA-PKcs or protein phosphatase-treated samples were resolved by 7 or 8% SDS-PAGE and stained with Coomassie Blue R-250. SV40 T-antigen was used as a sample loading control and was detected with an antiSV40 T-Ag antibody (Santa Cruz Biotechnology, Santa Cruz, CA)
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