Cyclobutane pyrimide dimers (CPD) in DNA, which are formed when DNA is exposed to ultraviolet irradiation, block replication if the systems designed to repair the CPD sites fail. The repair enzyme T4 endonuclease V (T4 endo V) eliminates CPD damages. 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 ~60°. We have developed single molecule fluorescence (SMF) and fluorescence resonance energy transfer (FRET) spectroscopy approaches to study the repair mechanism and kinetics of T4 endo V in real time. The SMF experiment is done using a Luca-Andor EM camera and a Nikon microscope. Micro-Manager, an open-source software that was modified to permit the monitoring of intensity in two regions of interest, in real time. We have modified the software to produce an accessible data file with which the recorded data can be analyzed proceeding the experiment. As a proof of principle, the optical setup built for the SMF experiments and the software was tested using quantum dots to prove the efficiency of the equipment in imaging and monitoring particles approaching the picometer scale and these results will be presented. To study the bending mechanism of CPD damaged DNA we used FRET spectroscopy. The FRET spectroscopy was done by using a Cy3 donor fluorophore and a Cy5 acceptor fluorophore on both damaged and undamaged DNA.Compared to the crystal structure of the T4 endo V-DNA complex, Cy3 and Cy5 fluorophores were strategically localized to use FRET to study the bent conformation. We have characterized the FRET properties using SMF, steady-state measurements, and gel electrophoresis. The decrease of the Cy3 fluorophore and increase of the Cy5 from the emission intensities indicates that FRET is occurring between both fluorophores. With FRET occurring, the steady state and SMF experimentshas helped us to understand the bending of DNA in CPD damaged DNA. This project is important because it will lead to a better understanding of how T4 endo V finds damaged sites as well as how, when, and why the structure of the DNA is distorted during the repair process.

Development of an undergraduate-friendly single-molecule fluorescence system to study DNA base flipping and protein-DNA-interaction in UV-damaged DNA

255th National Meeting (2018)

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