Journal of Biological Chemistry
Volume 279, Issue 38, 17 September 2004, Pages 39408-39413
Journal home page for Journal of Biological Chemistry

DNA: Replication, Repair, and Recombination
Non-homologous End Joining Requires That the DNA-PK Complex Undergo an Autophosphorylation-dependent Rearrangement at DNA Ends*

https://doi.org/10.1074/jbc.M406432200Get rights and content
Under a Creative Commons license
open access

Repair of chromosome breaks by non-homologous end joining requires the XRCC4-ligase IV complex, Ku, and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). DNA-PKcs must also retain kinase activity and undergo autophosphorylation at six closely linked sites (ABCDE sites). We describe here an end-joining assay using only purified components that reflects cellular requirements for both Ku and kinase-active DNA-PKcs and investigate the mechanistic basis for these requirements. A need for DNA-PKcs autophosphorylation is sufficient to explain the requirement for kinase activity, in part because autophosphorylation is generally required for end-joining factors to access DNA ends. However, DNA-PKcs with all six ABCDE autophosphorylation sites mutated to alanine allows access to ends through autophosphorylation of other sites, yet our in vitro end-joining assay still reflects the defectiveness of this mutant in cellular end joining. In contrast, mutation of ABCDE sites to aspartate, a phosphorylation mimic, supports high levels of end joining that is now independent of kinase activity. This is likely because DNA-PKcs with aspartate substitutions at ABCDE sites allow access to DNA ends while retaining affinity for Ku-bound ends and stabilizing recruitment of the XRCC4-ligase IV complex. Autophosphorylation at ABCDE sites thus apparently directs a rearrangement of the DNA-PK complex that ensures access to broken ends and joining steps are coupled together within a synaptic complex, making repair more accurate.

Cited by (0)

*

This work was supported by U. S. Public Health Service Grants CA-84442 (to D. A. R.) and AI32600 and AI42938 (to K. M.) and by the Alberta Heritage Foundation for Medical Research and the Canadian Institutes for Health Research (to the laboratory of S. P. L.-M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.