17-Hydroxyprogesterone While desolvation appears to play a u

While desolvation appears to play a universal role in the binding of the nucleotide substrate during TLS, the molecular forces regulating the polymerization step are more divergent as they depend upon the physical nature of the DNA lesion. For instance, π-stacking interactions play a large role in facilitating the polymerization step during the replication of non-instructional lesions such as abasic sites. This is based on the fact that artificial analogs such as 5-CITP and 5-MeCITP that possess significant π-electron density also display incredibly fast kpol values of 67s and 79s, respectively. In contrast, the rate constant for the polymerization step during the replication of the miscoding lesion, 8-oxo-G, depends more upon hydrogen-bonding interactions. This is evident as all of the nucleotide analogs tested here, which contain modifications to hydrogen-bonding groups, have lower kpol values compared to dATP. In fact, artificial analogs such as 5-CITP and 5-MeCITP that are incorporated opposite an abasic site with fast kpol values of ~70s are inserted opposite 8-oxo-G with lower kpol values of ~0.23s. Do other DNA polymerases utilize nucleobase desolvation during TLS? During chromosomal replication, high-fidelity DNA polymerases accurately and efficiently replicate undamaged DNA. In contrast, the activity of these DNA polymerases is significantly hindered when replicating damaged DNA. As a result, specialized DNA polymerases such as pol eta, pol kappa, and pol iota are recruited to participate more intimately in the efficient replication of unrepaired DNA lesions. However, the ability of specialized DNA polymerases to effectively perform TLS comes at a cost as they generally display reduced fidelity when replicating undamaged DNA. Current models attempting to explain this dichotomy are based primarily on structural differences that exist between the two 17-Hydroxyprogesterone of DNA polymerase [40], [41], [42]. In general, both high-fidelity and specialized DNA polymerases possess a similar global architecture that resembles a right hand and contains elements corresponding to fingers, palm, and thumb domains [43], [44]. However, close inspection reveals that the active sites of most specialized DNA polymerases are significantly larger than those of high-fidelity DNA polymerases. The expanded active site of specialized DNA polymerases is often used to explain how these polymerases can replicate large, bulky lesions, whereas the more constrained active site of high-fidelity polymerases hinders their ability to efficiently replicate damaged DNA. At face value, the results presented here using modified nucleotide analogs are consistent with the mechanism. However, we propose that nucleobase solvation also plays an important role in achieving nucleotide discrimination, especially during the replication of damaged DNA. An excellent example of this phenomenon comes from the kinetic studies here demonstrating that gp43 binds dATP very poorly when replicating 8-oxo-G. In this case, we propose that the weaker binding affinity reflects energetic penalties associated with stripping away water molecules that are bound to key hydrogen-bonding groups present on the natural nucleotide. The inference here is that the association of water molecules with these functional groups creates a solvation sphere around the nucleobase, which increases the overall size of the nucleotide. The resulting increase in size hinders efficient binding within the constrained active sites of high-fidelity polymerases. As demonstrated here, modifications such as alkylation that increase the overall hydrophobicity of the nucleobase also reduce the size of this solvation sphere. The biophysical consequence is that the smaller size of the nucleotide makes binding to the polymerase more efficient and lowers the energetic penalties required for complete desolvation of the incoming nucleotide.