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 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.
Highlights
Ku translocates inward on the DNA in an ATP-independent manner, forming the active DNA-PK complex [3, 4]
The Ku-DNA co-crystal structure reveals no direct contact with the nucleobases but contain a number of hydrophobic amino acids positioned within the major groove of the duplex DNA termini and basic amino acids that interact with the phosphate backbone [8]
We have identified a redox-sensitive conformational change in Ku and the effect that it has on the kinetics of the Ku-DNA interaction
Summary
Recent reports have demonstrated a link between redox stress and the NHEJ pathway. This connection has been demonstrated in cells where increased oxidative stress results in decreased DNA-PK activity and where decreased oxidative stress increased DNA-PK activity [17]. To further investigate the effect of redox on Ku structure and function, we employed the intrinsic fluorescence of Ku and the subsequent quenching that occurs upon binding to DNA to assess the kinetics of binding. In addition to defining the pre-steady state kinetic interaction of Ku with duplex DNA, we identified a mechanism of redox regulation of Ku binding and demonstrated that the regulation is dependent on a conformational change that influences the Ku-DNA fluorescence and dissociation rate
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