National Institutes of Health/National Cancer Institute (NIH/NCI)
CA100504
United States
National Institutes of Health/National Cancer Institute (NIH/NCI)
GM118009
United States
Citation
Journal: Nucleic Acids Res / Year: 2022 Title: Structural analysis of the basal state of the Artemis:DNA-PKcs complex. Authors: Go Watanabe / Michael R Lieber / Dewight R Williams / Abstract: Artemis nuclease and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) are key components in nonhomologous DNA end joining (NHEJ), the major repair mechanism for double-strand DNA breaks. ...Artemis nuclease and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) are key components in nonhomologous DNA end joining (NHEJ), the major repair mechanism for double-strand DNA breaks. Artemis activation by DNA-PKcs resolves hairpin DNA ends formed during V(D)J recombination. Artemis deficiency disrupts development of adaptive immunity and leads to radiosensitive T- B- severe combined immunodeficiency (RS-SCID). An activated state of Artemis in complex with DNA-PK was solved by cryo-EM recently, which showed Artemis bound to the DNA. Here, we report that the pre-activated form (basal state) of the Artemis:DNA-PKcs complex is stable on an agarose-acrylamide gel system, and suitable for cryo-EM structural analysis. Structures show that the Artemis catalytic domain is dynamically positioned externally to DNA-PKcs prior to ABCDE autophosphorylation and show how both the catalytic and regulatory domains of Artemis interact with the N-HEAT and FAT domains of DNA-PKcs. We define a mutually exclusive binding site for Artemis and XRCC4 on DNA-PKcs and show that an XRCC4 peptide disrupts the Artemis:DNA-PKcs complex. All of the findings are useful in explaining how a hypomorphic L3062R missense mutation of DNA-PKcs could lead to insufficient Artemis activation, hence RS-SCID. Our results provide various target site candidates to design disruptors for Artemis:DNA-PKcs complex formation.
Name: Y-shape duplex DNA / type: dna / ID: 3 Details: This oligo forms Y-shape double hairpins at the closed end and an open pseudo-Y-shape with short overhangs at the open end. Classification: DNA
Cryogen name: ETHANE / Chamber humidity: 73 % / Chamber temperature: 295 K Details: Plunge-freeze was performed using a home-made manual plunger at typical indoor humidity (Los Angeles, CA) and at room temperature..
Details
This sample was monodisperse.
-
Electron microscopy
Microscope
FEI TITAN KRIOS
Specialist optics
Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recording
Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Average exposure time: 3.0 sec. / Average electron dose: 45.0 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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