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

Morris, E. P., Rivera-Calzada, A., da Fonseca, P. C. A., Llorca, O., Pearl, L. H. and Spagnolo, L. (2011) Evidence for a remodelling of DNA-PK upon autophosphorylation from electron microscopy studies. Nucleic Acids Research, 39(13), pp. 5757-5767. (doi: 10.1093/nar/gkr146) (PMID:21450809) (PMCID:PMC3141256)

Abstract

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: Articles
• Additional Information: The Spanish Ministry of Science and Innovation (SAF2008-00451 to O.L.); The Human Frontiers Science Program (RGP39/2008 to O.L.); Red Tema´tica Investigacio´n Cooperativa en Ca´ncer (RD06/0020/1001 to O.L.); Programme Grant from Cancer Research UK (to L.H.P.); The Spanish National Research Council, The European Social Fund (A.R.-C.); Cancer Research UK Project Grant (C17335/A10470 to L.S.). Funding for open access charge: Cancer Research UK Project (Grant C17335/A10470).
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Spagnolo, Professor Laura
• Authors: Morris, E. P., Rivera-Calzada, A., da Fonseca, P. C. A., Llorca, O., Pearl, L. H., and Spagnolo, L.
• College/School: College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
• Journal Name: Nucleic Acids Research
• Publisher: Oxford University Press
• ISSN: 0305-1048
• ISSN (Online): 1362-4962
• Copyright Holders: Copyright: © The Author(s) 2011
• First Published: First published in Nucleic Acids Research 39(13): 5757-5767
• Publisher Policy: Reproduced under a Creative Commons licence