Understanding the factors that control the fidelity of DNA polymerases is a problem of great fundamental andpractical importance. Here we propose to exploit our recent advances and to continue the use of computersimulation approaches to gain a deeper insight that will complement the experimental progress in projects 1and 3. It is proposed to use reliable theoretical tools for structure/fidelity correlation and to refine thiscorrelation by a constant feedback from kinetic, binding, and structural experiments. The proposed projectsinclude: (i) Establishing the cleavage mechanism of the PaO bond of dNTP substrates in the polymeraseactive site by a concerted use of different theoretical approaches, ranging from ab initio QM-MM free energycalculations to constraint DFT free energy calculations and to systematic empirical valence bond studies, (ii)Refining our calculations of substrate binding free energies as well as calculations of the effects of pol pimutations on the these binding energies, (iii) Exploring factors that control the fidelity of polymerases while|paying special attention to the allosteric transfer of information between the base binding site and the!chemical reaction site. The allosteric information transfer will be studied by calculating the coupling betweendifferent residues and the transition state (TS) using the correlation matrix formalism. (iv)Exploring the natureof the TS for incorporating right (R) and wrong (W) nucleotides, including the study of the effect of theprotein conformational landscape on the nature of the TS. These studies will be helped by structural studiesof transition state analogues (TSAs) with correct and incorrect base pairs, (v) Determining the structure andbinding energy of different TSAs that will be examined experimentally by our coworkers (Project 3) andexplored theoretically. This study will teach us about the relationship between the TSAs and the actual TSand will help in verifying the calculated TS properties.All the above studies will involve constant feedback from the experimental counterparts of the project. Thiscollaboration will help us develop a more general and coherent view about the nature of DNA polymerasefidelity.
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