DNA-PKcs dependence of Artemis endonucleolytic activity, differences between hairpins and 5' or 3' overhangs.
During V(D)J recombination, the RAG proteins create DNA hairpins at the V, D, or J coding ends, and the structure-specific nuclease Artemis is essential to open these hairpins prior to joining. Artemis also is an endonuclease for 5' and 3' overhangs at many DNA double strand breaks caused by ionizing radiation, and Artemis functions as part of the nonhomologous DNA end joining pathway in repairing these. All of these activities require activation of the Artemis protein by interaction with and phosphorylation by the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). In this study, we have identified a region of the Artemis protein involved in the interaction with DNA-PKcs. Furthermore, the biochemical and functional analyses of C-terminally truncated Artemis variants indicate that the hair-pin opening and DNA overhang endonucleolytic features of Artemis are triggered by DNA-PKcs in two modes. First, autoinhibition mediated by the C-terminal tail of Artemis is relieved by phosphorylation of this tail by DNA-PKcs. Thus, C-terminally truncated Artemis derivatives imitate DNA-PKcs-activated wild type Artemis protein and exhibit intrinsic hairpin opening activity. Second, DNA-PKcs may optimally configure 5' and 3' overhang substrates for the endonucleolytic function of Artemis.
Niewolik
Doris
D
Institute for Clinical Transfusion Medicine and Immunogenetics and the Department of Transfusion Medicine, the University Hospital Ulm, D-89081 Ulm, Germany.
Pannicke
Ulrich
U
Lu
Haihui
H
Ma
Yunmei
Y
Wang
Ling-Chi Vicky
LC
Kulesza
Peter
P
Zandi
Ebrahim
E
Lieber
Michael R
MR
Schwarz
Klaus
K
D000818:Animals; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage; D004260:DNA Repair; D051747:DNA-Activated Protein Kinase / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D004720:Endonucleases; D006801:Humans; D015151:Immunoblotting; D007668:Kidney / Q000378:metabolism; D013058:Mass Spectrometry; D008954:Models, Biological; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011839:Radiation, Ionizing; D011995:Recombination, Genetic; D045567:VDJ Recombinases / Q000378:metabolism
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D004247:DNA; D051747:DNA-Activated Protein Kinase; D045567:VDJ Recombinases; C426344:DCLRE1C protein, human; D004706:Endodeoxyribonucleases; D004720:Endonucleases