DNA-dependent conformational changes in the Ku heterodimer

Jason A. Lehman, Derek J. Hoelz, John Turchi

Research output: Contribution to journalArticle

27 Citations (Scopus)

Abstract

Ionizing radiation induces DNA double-strand breaks which are repaired by the nonhomologous end joining (NHEJ) pathway. NHEJ is initiated upon Ku binding to the DNA ends and facilitating an interaction with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). This heterotrimeric DNA-PK complex is then active as a serine/threonine protein kinase. The molecular mechanisms involved in DNA-PK activation are unknown. Considering the crucial role of Ku in this process, we therefore determined the influence of DNA binding on the structure of the Ku heterodimer. Chemical modification with NHS-biotin and mass spectrometry were used to identify sites of modification. Biotinylation of free Ku revealed several reactive lysines on Ku70 and Ku80 which were reduced or eliminated upon DNA binding. Interestingly, in the predicted C-terminal SAP domain of Ku70, biotinylation patterns were observed which suggest a structural change in this region of the protein induced by DNA binding. Limited proteolytic digests of free and DNA-bound Ku revealed a series of unique peptides, again, indicative of a change in the accessibility of the Ku70 and Ku80 C-terminal domains. A 10 kDa peptide was also identified which was preferentially generated under non-DNA-bound conditions and mapped to the C-terminus of Ku70. These results indicate a DNA-dependent movement or structural change in the C-terminal domains of Ku70 and Ku80 that may contribute to DNA-PKcs binding and activation. These results represent the first demonstration of DNA-induced changes in Ku structure and provide a framework for analysis of DNA-PKcs and the mechanism of DNA-PK activation.

Original languageEnglish
Pages (from-to)4359-4368
Number of pages10
JournalBiochemistry
Volume47
Issue number15
DOIs
StatePublished - Apr 15 2008

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DNA
DNA-Activated Protein Kinase
Biotinylation
Catalytic Domain
Chemical activation
Joining
Ku Autoantigen
Peptides
Double-Stranded DNA Breaks
Protein-Serine-Threonine Kinases
DNA-Binding Proteins
Ionizing Radiation
Lysine
Ionizing radiation
Chemical modification
Mass Spectrometry
Mass spectrometry
Demonstrations
Proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

DNA-dependent conformational changes in the Ku heterodimer. / Lehman, Jason A.; Hoelz, Derek J.; Turchi, John.

In: Biochemistry, Vol. 47, No. 15, 15.04.2008, p. 4359-4368.

Research output: Contribution to journalArticle

Lehman, Jason A. ; Hoelz, Derek J. ; Turchi, John. / DNA-dependent conformational changes in the Ku heterodimer. In: Biochemistry. 2008 ; Vol. 47, No. 15. pp. 4359-4368.
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