DNA-PKcs Dependence of Artemis Endonucleolytic Activity, Differences between Hairpins and 5′ or 3′ Overhangs

Doris Niewolik,Ulrich Pannicke,Haihui Lu,Yunmei Ma,Ling-Chi Vicky Wang,Peter Kulesza,Ebrahim Zandi,Michael R Lieber,Klaus Schwarz

doi:10.1074/jbc.m606023200

Doris Niewolik, Ulrich Pannicke + Show 7 more

Open Access

https://doi.org/10.1074/jbc.m606023200

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Abstract

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.

Highlights

V(D)J4 recombination is the somatic recombination in precursor lymphocytes by which functional and highly diverse immunoglobulin and T cell receptor genes are generated [1,2,3]
A defect in either RAG-1/-2, Artemis, DNA ligase IV, or XLF/Cernunnos leads to the phenotypes of severe combined immunodeficiency, Omenn syndrome, or other immunodeficiencies with perturbed lymphocyte development, because of the impaired generation of immunoglobulin and T cell receptor genes
We found that C-terminal deletion of amino acids up anti-mouse IgG-fluorescein isothiocyanate for 1 h at room tempera- Artemis protein with DNA-PKcs (Fig. 1)

Summary

EXPERIMENTAL PROCEDURES

Protein Expression Constructs—Human wild type Artemis expression plasmids were constructed as described [23]. The expression plasmid pPK1 encoding full-length human DNA-PKcs was constructed as described [26]. Human primary Artemis-negative fibroblasts were transfected using AMAXA NHDF-Neo nucleofector kit (AMAXA Biosystems, Cologne, Germany) as described [23]. Myctagged DNA-PKcs and Myc-tagged Artemis were immunoprecipitated and detected with anti-Myc antibody (Invitrogen). Immunostaining of AA8 and V-3 Cells—The Chinese hamster ovary cell lines AA8 (DNA-PKcs-positive) and V-3 (DNAPKcs-negative) were transfected by nucleofection using the AMAXA nucleofector kit T (AMAXA Biosystems) with empty pcDNA6/myc-His version A or plasmid coding for wild type or mutant forms of Artemis. The relative survival advantage was calculated as percentage of green cells in irradiated samples divided by the percentage of green cells in unirradiated samples This ratio was normalized to the corresponding value obtained with the control (only EGFP expressing) vector. Each sample was analyzed by two experiments using different scan modes, a general MS/MS scan mode, and an MS3 data-dependent neutral loss mode for specific detection of phosphopeptides

Are Important for Interaction with

DISCUSSION

The Endonucleolytic Activities on