Fluorescence Approach to Study Base Flipping in the DNA Repair Mechanism of T4 Endonuclease V

Abstract

UV irradiation can cause the formation of cyclobutane pyrimide dimers (CPD), which are the most common type of DNA damage that can lead to the development of skin cancer. However, living organisms have proteins to repair DNA, and that perform a base-flipping process to recognize, gain access to, and repair damaged nucleotides. Different ensemble-average experiments have been developed in order to understand the base-flipping process. However, ensemble averaged measurements can mask dynamic fluctuations in the formation of intermediate complexes and lead to different interpretations of the repair process.T4 endonuclease V (T4-endo-V) is a repair enzyme that eliminates CPD damages. The static crystal structure of T4-endo-V has demonstrated that the base-flipping process is part of the mechanism used in the repair work. In the absence of a more sophisticated method to monitors protein-DNA binding, DNA structural changes and base-flipping simultaneously, the interpretation of the formation of intermediates and the importance of base-flipping during the DNA repair process remains ambiguous.Single molecule fluorescence can be used to determine the formation of one or more intermediates and also to study the kinetics of the mechanisms in enzymatic reactions. In our single molecule fluorescence approach, we will use the fluorescent base analog 6MAP to probe the base flipping state and FRET to study the T4-endo-V-DNA binding mechanism. We have characterized the steady-state fluorescence properties of different DNA constructions under different physiological conditions to demonstrate the stability of our base-flipping probe. In addition, we have performed FRET measurements in CPD-damaged-DNAs to study global conformational changes in the DNA structure. With this approach we will be able to fully understand how T4-endo-V uses base-flipping to recognize and repair damaged DNA.We gratefully acknowledge financial support from CSUPERB and NSF (1237548).