UV light from the sun or artificial sources cause the formation of cyclobutane pyrimide dimers (CPD), which can result in mutations that could block replication if the systems designed to repair this damage fail. T4 endonuclease V (T4 endo V) is a bacterial repair enzyme that eliminates CPD damage. The crystal structure of T4 endo V shows that when the protein complexes with a double helix DNA containing a thymine dimer, the 5’ side complementary adenine flips out and makes the dimer accessible for the catalytic reaction without affecting the adenine base. The crystal structure also shows the DNA is bent ~60o. However, the static crystal structure of T4 endo V has only demonstrated that the bending process is part of the mechanism used in the repair work to gain access to the target site. It is not clear if the enzyme finds the damage by scanning the DNA (sliding) or by direct binding to the canonical site. In addition, it is not well understood whether the enzyme induces the DNA kink after finding the damaged site (active mechanism), or if the kink corresponds to the recognition mechanism of the damaged site before binding (passive mechanism). We have developed single molecule fluorescence (SMF) approaches to study the repair mechanism and kinetics of T4 endo V in real time. SMF spectroscopy is the most powerful technique, and the only direct method, to analyze the formation of intermediates and biological complexes in real time that are normally measured in ensemble experiments. We have developed single molecule assays to monitor the base flipping mechanism in the DNA-T4 endo V complex in real time. To accomplish this goal, we studied the fluorescent base analog 6MAP to probe base flipping. To study the bending mechanism of CPD damaged DNA we used single molecule fluorescence resonance energy transfer (FRET). We have also characterized the fluorescence properties (steady-state and TCSPC) of 6MAP and its dependence in different surroundings, such as different Mg2+ and Na+ concentrations, and different pH conditions. Compared to the crystal structure of the T4 endo V-DNA complex, fluorophores have been strategically localized to use FRET to study the bent conformation. We have characterized the FRET properties using SMF, steady-state measurements, and gel electrophoresis. This project will lead to a better understanding of how T4 endo V finds damaged sites; and how, when, and why the structure of the DNA is distorted during the repair process.

Single molecule approach to study the repair mechanism of T4 endo V

251st National Meeting (2016)

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