Human DNA polymerase eta (Pol eta) plays a vital role in protection against skin cancer caused by damage from ultraviolet light. This enzyme rescues stalled replication forks at cyclobutane thymine-thymine dimers (TTDs) by inserting nucleotides opposite these DNA lesions. Residue R61 is conserved in, the Pol eta enzymes across. species, but the corresponding residue; as well as its neighbor S62, is different in other Y-fainily polymerases, Pol l and Pol kappa. Herein, R61 and S62 are mutated to their Pol l and Pol kappa counterparts. Relative binding free energies of dATP to mutant Pol eta center dot DNA. complexes with and without a TTD) were calculated using thermodynamic integration. The binding free energies of dATP to the Pol eta center dot DNA complex with and without a TTD are more similar for all of these mutants than for wild-type Pol eta, suggeting that these mutations decrease the ability if this enzyme to distinguish between a TTD lesion and undamaged DNA. Molecular dynamics simulations of the mutant systems provide insights into the molecular level basis for the changes in relative binding free energies. The simulations identified differences in hydrogen-bonding, cation-pi, and pi-pi interactions of the side chains with the dATP and the TTD or thymine-thymine (TT) motif. The simulations also revealed that R61 and Q38 act as a clamp to position the dATP and the TTD Or TT and that the mutations impact the balance among the interactions related to this clamp. Overall, these calculations suggest that R61 and S62 play key roles in the specificity and effectiveness of Pol eta for bypassing TTD lesions during DNA replication. Understanding the basis for this specificity is important for designing drugs aimed at canter treatment.