Kinetic analysis of the Ku-DNA binding activity reveals a redox-dependent alteration in protein structure that stimulates dissociation of the Ku-DNA complex.
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 a model 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.
Andrews
Brooke J
BJ
Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana 46202.
Lehman
Jason A
JA
Department of Biomedical Sciences, Wright State University, Dayton, Ohio 45435.
Turchi
John J
JJ
Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana 46202; Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202. Electronic address: jturchi@iupui.edu.
D004247:DNA / Q000378:metabolism*; D004265:DNA Helicases / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D000072200:Ku Autoantigen; D010084:Oxidation-Reduction; D011485:Protein Binding; D011487:Protein Conformation
D004247:DNA; D004265:DNA Helicases; C106893:XRCC5 protein, human; D000072200:Ku Autoantigen