Kinetic analysis of the Ku-DNA binding activity reveals a redox-dependent alteration in protein structure that stimulates dissociation of the Ku-DNA complex

Brooke J. Andrews, Jason A. Lehman, John Turchi

Research output: Contribution to journalArticle

35 Citations (Scopus)

Abstract

Ku is a heterodimeric protein comprising 70- and 80-kDa subunits that participate in the non-homologous end-joining (NHEJ) repair pathway for rejoining DNA double strand breaks. We have analyzed the pre-steady state binding of Ku with various DNA duplex substrates and identified a redox-sensitive Ku-DNA interaction. Pre-steady state analysis of Ku DNA binding was monitored via intrinsic Ku quenching upon binding DNA and revealed that, under fully reduced conditions, binding occurred in a single-step process. Reactions performed under limited reduction revealed a two-step binding process, whereas under fully oxidized conditions, we were unable to detect quenching of Ku fluorescence upon binding DNA. The differential quenching observed under the different redox conditions could not be attributed to two Ku molecules binding to a single substrate or Ku sliding inward on the substrate. Although only modest differences in Ku DNA binding activity were observed in the stoichiometric anisotropy and electrophoretic mobility shift assay studies, as a function of redox conditions, a dramatic difference in the rate of Ku dissociation from DNA was observed. This effect was also induced by diamide treatment of Ku and could be abrogated by dithiothreitol treatment, demonstrating a reversible redox effect on the stability of the Ku-DNA complex. The redox-dependent alteration in Ku-DNA interactions is manifested by a redox-dependent alteration in Ku structure, which was confirmed by limited proteolysis and mass spectrometry analyses. The results support amodel for the interaction of Ku with DNA that is regulated by redox status and is achieved by altering the dissociation of the Ku-DNA complex.

Original languageEnglish
Pages (from-to)13596-13603
Number of pages8
JournalJournal of Biological Chemistry
Volume281
Issue number19
DOIs
StatePublished - May 12 2006

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Oxidation-Reduction
Kinetics
DNA
Proteins
Quenching
Substrates
Diamide
Proteolysis
Double-Stranded DNA Breaks
Electrophoretic mobility
Dithiothreitol
Anisotropy
Electrophoretic Mobility Shift Assay
Joining
Mass Spectrometry
Mass spectrometry
Assays
Fluorescence
Repair
Molecules

ASJC Scopus subject areas

  • Biochemistry

Cite this

Kinetic analysis of the Ku-DNA binding activity reveals a redox-dependent alteration in protein structure that stimulates dissociation of the Ku-DNA complex. / Andrews, Brooke J.; Lehman, Jason A.; Turchi, John.

In: Journal of Biological Chemistry, Vol. 281, No. 19, 12.05.2006, p. 13596-13603.

Research output: Contribution to journalArticle

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