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TitleStructural analysis of the basal state of the Artemis:DNA-PKcs complex.
Journal, issue, pagesNucleic Acids Res, Vol. 50, Issue 13, Page 7697-7720, Year 2022
Publish dateJul 22, 2022
AuthorsGo Watanabe / Michael R Lieber / Dewight R Williams /
PubMed AbstractArtemis 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.
External linksNucleic Acids Res / PubMed:35801871 / PubMed Central
MethodsEM (single particle)
Resolution3.33 - 3.77 Å
Structure data

26192

7tyr

EMDB-26192: Cryo-EM structure of the basal state of the Artemis:DNA-PKcs complex
PDB-7tyr: Cryo-EM structure of the basal state of the Artemis:DNA-PKcs complex (see COMPND 13/14)
Method: EM (single particle) / Resolution: 3.33 Å

EMDB-26198: Cryo-EM structure of the Artemis:DNA-PKcs:DNA complex
Method: EM (single particle) / Resolution: 3.77 Å

Source
  • homo sapiens (human)
  • synthetic construct (others)
KeywordsDNA BINDING PROTEIN / Kinase / nuclease

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