In the case of high fidelity polymerases we

In the case of high-fidelity polymerases, we propose that water is used to achieve negative selection against nucleotide binding. With specialized DNA polymerases, we propose that nucleobase desolvation plays a different yet important role in allowing these enzymes to replicate damaged DNA. In this model, specialized DNA polymerases use enthalpic–entropic compensation as a way to generate high catalytic efficiency during TLS. This is possible as the expanded active site of a specialized AG-1295 is large enough to bind a fully solvated nucleotide. This essentially bypasses the initial requirement for nucleobase desolvation. Consistent with this mechanism are previous data obtained using the specialized DNA polymerase, pol η, in which the Km values measured for modified and artificial nucleotides were independent of their hydrophilic or hydrophobic nature [37]. For instance, the Km for dATP is 47μM, while the Km values for hydrophobic analogs such as N6-Me-dATP, O6-Me-dGTP, 5-EyITP, and 5-NITP remain invariant at ~50μM [37]. While this mode of binding may seem counterproductive, the ability of specialized DNA polymerases to bind a fully solvated nucleotide could play two important roles in its primary function to replicate damaged DNA. First, water molecules surrounding the incoming nucleobase could move within the active site of the polymerase. This mobility could generate greater flexibility that could subsequently provide these DNA polymerases opportunities to optimize productive interactions between the incoming nucleotide and a DNA lesion. In this model, the greater entropy provided by increased water mobility may allow these polymerases to accommodate a variety of structurally distinct DNA lesions ranging from non-instructional including abasic sites to crosslinked lesions such as thymine dimers and cisplatinated DNA. In addition, the larger solvation sphere could also provide enthlapic stabilization by increasing hydrogen-bonding interactions that are required for interactions between the incoming nucleotide and a DNA lesion. The synergy between these features may account for improved efficiency of specialized polymerases such as pol eta to perform TLS. In addition, this may also explain why certain specialized DNA polymerases display reduced fidelity when replicating undamaged DNA. In this case, the ability of the polymerase to move water molecules within its active site could allow the polymerase to form mispairs more easily. While the kinetic studies described here provide initial evidence redefining the role of nucleobase desolvation during replication, we acknowledge that more experimentation is needed to fully quantify this biophysical feature during normal and translesion DNA synthesis. Toward this goal, we are currently examining the effects of molecular crowding agents and solvent isotope effects to further explore the role of nucleobase desolvation by both high-fidelity and specialized DNA polymerases.
Material and Methods