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Abstract
Here, we report the study of a new multichannel DNA fluorescent base analogue 3-hydroxychromone (3HC) to evaluate its suitability as a fluorescent reporter probe of structural transitions during protein-DNA interactions and its comparison with the current commercially available 2-aminopurine (aPu), pyrrolocytosine (Cpy) and 1,3-diaza-2-oxophenoxazine (tCO). For this purpose, fluorescent base analogues were incorporated into DNA helix on the opposite or on the 5′-side of the damaged nucleoside 5,6-dihydrouridine (DHU), which is specifically recognized and removed by Endonuclease VIII. These fluorophores demonstrated different sensitivities to the DNA helix conformational changes. The highest sensitivity and the most detailed information about the conformational changes of DNA induced by protein binding and processing were obtained using the 3HC probe. The application of this new artificial fluorescent DNA base is a very useful tool for the studies of complex mechanisms of protein-DNA interactions. Using 3HC biosensor, the kinetic mechanism of Endonuclease VIII action was specified.
Highlights
Understanding at the molecular level the dynamics and functions of enzymes in interactions with their DNA targets is of main importance in biology and medicine
The minimal kinetic scheme describing the observed changes of Trp fluorescence intensity was identical to scheme in Figure 3 and contained three equilibrium steps that characterized substrate binding followed by an irreversible chemical step and an equilibrium step of product release
The formation of enzyme with the reaction product (ENP) complex in the chemical step registered by Trp fluorescence changes is in good agreement with the accumulation of the reaction products detected by PAGE analysis (Fig. 7B)
Summary
Understanding at the molecular level the dynamics and functions of enzymes in interactions with their DNA targets is of main importance in biology and medicine. In this context, fluorescence spectroscopy is widely used in nucleic acids research to study the structure and the dynamics as well as the kinetics of protein-DNA interactions [1]. The sensitivity to nucleobase quenching has been exploited to study in real time the dynamic and function of nucleic acids. The most commonly used fluorescent base analogue is 2-aminopurine (aPu, Fig. 1). The sensitivity of aPu fluorescence to the microenvironment has been utilized in the study of wide range protein-DNA interactions [10,11,12,13,14]. The cytosine analogue pyrrolocytosine (3-[b-D-2-ribofuranosyl]-6-methylpyrrolo[2,3-d]pyrimidin-2(3H)-one, Cpy, Fig. 1)
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