On the Origin of Sugar Selectivity by DNA Polymerases

Abstract

Gene replication and transcription are the core biological processes. These processes are catalyzed by DNA and RNA polymerases. These two types of polymerases show different selectivity on the substrate based on the presence of an extra 2’ hydroxyl group on the ribose sugar. DNA polymerases adopt deoxyribose nucleotides (dNTP) while RNA polymerases adopt ribose nucleotides (rNTP). Currently, the general consensus for the sugar discrimination mechanism observed in many nucleotide polymerases is that the highly conserved tyrosine/phenylalanine residues play a critical role. For example, Y416 in RB69 DNA polymerase blocks the bulkier 2’ OH on the ribose sugar in rNTP [Yang et al. 2002 Biochem]. However, it is often easy for proteins to avoid such steric clashes by moving the associated residues away which does not cost a lot of energy. Such hypothesis can be explicitly tested by calculating the actual energetic terms. In this study, we examined the steric gate hypothesis by free energy perturbation (FEP) calculation based on alchemistic mutational pathway from rNTP to dNTP in RB69 DNA polymerase. As a result, our FEP calculation does support the steric gate mechanism although we also observes a moderate electrostatic contributions as well. Interestingly, the distance between 2’ OH and the side chain of the Y416 during our simulation was further than observed in the original structure which alleviates the exaggerated steric clash. We will also use this opportunity to introduce our water flooding approach which improved our results as a result of using better structures with efficiently and optimally inserted water molecules in the protein site.