Evidence for a remodelling of DNA-PK upon autophosphorylation from electron microscopy studies

Morris, Edward P, Rivera-Calzada, Angel, da Fonseca, Paula C A, Llorca, Oscar, Pearl, Laurence H and Spagnolo, Laura (2011) Evidence for a remodelling of DNA-PK upon autophosphorylation from electron microscopy studies. Nucleic Acids Research, 39 (13). pp. 5757-5767. ISSN 1362-4962

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The multi-subunit DNA-dependent protein kinase (DNA-PK), a crucial player in DNA repair by non-homologous end-joining in higher eukaryotes, consists of a catalytic subunit (DNA-PKcs) and the Ku heterodimer. Ku recruits DNA-PKcs to double-strand breaks, where DNA-PK assembles prior to DNA repair. The interaction of DNA-PK with DNA is regulated via autophosphorylation. Recent SAXS data addressed the conformational changes occurring in the purified catalytic subunit upon autophosphorylation. Here, we present the first structural analysis of the effects of autophosphorylation on the trimeric DNA-PK enzyme, performed by electron microscopy and single particle analysis. We observe a considerable degree of heterogeneity in the autophosphorylated material, which we resolved into subpopulations of intact complex, and separate DNA-PKcs and Ku, by using multivariate statistical analysis and multi-reference alignment on a partitioned particle image data set. The proportion of dimeric oligomers was reduced compared to non-phosphorylated complex, and those dimers remaining showed a substantial variation in mutual monomer orientation. Together, our data indicate a substantial remodelling of DNA-PK holo-enzyme upon autophosphorylation, which is crucial to the release of protein factors from a repaired DNA double-strand break.

Item Type: Article
Schools and Departments: School of Life Sciences > Sussex Centre for Genome Damage and Stability
Subjects: Q Science > QD Chemistry > QD0241 Organic chemistry > QD0415 Biochemistry
Q Science > QH Natural history > QH0001 Natural history (General) > QH0201 Microscopy
Depositing User: Laurence Pearl
Date Deposited: 30 Sep 2014 14:35
Last Modified: 01 Jul 2019 15:17
URI: http://sro.sussex.ac.uk/id/eprint/50440

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