Damage to DNA has been implicated in numerous human diseases, particularly cancer, and the aging process. Our long term goal is to determine dynamic and structural information from damaged DNA and associated repair enzymes, and apply this information to understanding the fundamental aspects of DNA repair. Our specific hypothesis contains three parts: i) damage to DNA bases alters the local conformational dynamics, ii) these dynamics can be modeled and quantitatively correlated with repair enzyme kinetics and thermodynamics, and iii) the local dynamics play a role in the damage recognition process. This hypothesis is based on an established body of work achieved to determine how DNA is repaired. A process called base excision repair (BER) has evolved using glycosylases to help maintain DNA integrity. Glycosylase activity in BER contains several steps including 1) identifying the DNA damage, 2) forming an active enzyme-DNA complex, 3) removing the damaged base, and 4) removing the abasic site to allow for DNA polymerization. During the complexation and removal steps in BER, the damaged nucleotide is completely rotated out of the DNA helix and stabilized within the binding pocket of the glycosylase before the glycosidic bond is cleaved. This process is often referred to as base flipping and is a common motif in many protein-DNA interactions. The final three steps in BER are well characterized; however, the specific modes by which the repair enzymes identify the DNA damage and flip the damaged nucleotide remain unclear. Deformation of the local DNA structure during the binding and base flipping processes, which often also includes local DNA bending or reciprocal flipping of the nucleotide opposite the lesion, occurs at a significant energy cost that may be partially alleviated by local conformation flexibility (exhibited as large amplitude dynamics) at the lesion site. The proposal herein has two primary specific aims. First, local conformational dynamics in damaged free DNA will be characterized using deuterium solid-state nuclear magnetic resonance (SSNMR), and their biological role evaluated. Second, the local DNA dynamics in complex with a pyrimidine dimer glycosylase will be monitored via deuterium SSNMR.
The specific aims are designed to determine fundamental properties of damaged DNA, and the results determined will have implications in cancer research by helping determine essential aspects of DNA repair.
Cancer very often originates from damage to DNA, and knowledge of the fundamental aspects of how DNA damage is repaired will allow for better opportunities to find cures. This project proposes two avenues of research: i) to study flexibility of damaged free DNA and ii) study the changes in flexibility in damaged DNA bound to a repair enzyme. The work will determine if the flexibility plays a role in the repair process, and aid in the fundamental understanding of how cancer is prevented. ? ? ?
| Kinde-Carson, Monica N; Ferguson, Crystal; Oyler, Nathan A et al. (2010) Solid state 2H NMR analysis of furanose ring dynamics in DNA containing uracil. J Phys Chem B 114:3285-93 |
