Effects of Active Site Mutations on Specificity of Nucleobase Binding in Human DNA Polymerase η

Abstract

Human DNA polymerase η (Pol η) 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 η enzymes across species, but the corresponding residue, as well as its neighbor S62, is different in other Y-family polymerases, Pol ι and Pol κ. Herein, R61 and S62 are mutated to their Pol ι and Pol κ counterparts. Relative binding free energies of dATP to mutant Pol η•DNA complexes with and without a TTD were calculated using thermodynamic integration. The binding free energies of dATP to the Pol η•DNA complex with and without a TTD are more similar for all of these mutants than for wild-type Pol η, suggesting that these mutations decrease the ability of 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-π, and π-π 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 η for bypassing TTD lesions during DNA replication. Understanding the basis for this specificity is important for designing drugs aimed at cancer treatment.

Scheme 1. Thermodynamic Cycle Used for Calculating Relative Binding Free Energies of dATP to a Complex of Mutated Pol η and TTD- or TT-Containing DNA

Figure 1

WC base pairing for the dATP and the 3′T of TTD. “1” is defined as the hydrogen bond in which the 3′T(N3) of the TTD/TT is the donor heavy atom, the hydrogen attached to it is the donor hydrogen, and the dATP(N1) is the acceptor. “2” is defined as the hydrogen bond in which the dATP(N6) is the donor heavy atom, the HN2 attached to it is the donor hydrogen, and the 3′T(O4) of the TTD/TT is the acceptor.

Figure 2

Relative positioning of dATP and residue 61 in the WT and R61K systems with a TTD (a) or a TT (b) motif. The WT R61 is shown in gray shades, and the mutant K61 is shown in burgundy shades, along with their matching-colored labels. For both R61 and K61, one configuration is depicted at atomic level in gray and burgundy, respectively, and the other two configurations are depicted as sticks that are oriented along the side chain. In part b, two dominant configurations of the 5′T of the TT motif are shown and are labeled as “5′T(1)” and “5′T(2)”, where 5′T(2) is shown in blue shades. The R61 and K61 side chains adopt the conformations shown as sticks and at atomic level with the 5′T(1) conformation, while only the conformations shown as sticks are observed with the 5′T(2) conformation. The varying conformations of R61 were also observed in previous simulations.

Figure 3

Hydrogen-bonding (black dashed lines), π–π (orange dashed lines), and cation−π (pink dashed lines) interactions of R61 or K61 with dATP and TTD. Each frame shows the WC base pairing interactions between the dATP and the 3′T of TTD as black dashed lines. (a) The guanidinium side chain of R61 interacts with one of the α-phosphate oxygens through charge–charge and hydrogen-bonding interactions (black dashed line, circled). This configuration displays a T-shaped π–π (not indicated here) and a cation−π (pinked dashed line) interaction between the R61 side chain and dATP(N7). (b) The guanidinium side chain of R61 interacts with the dATP base through π–π (orange dashed lines) and cation−π (pink dashed line, virtually indistinguishable from orange lines) interactions. This side chain configuration of R61 also allows hydrogen-bonding interactions with the 5′T of TTD (black dashed lines). (c) The guanidinium side chain of R61 interacts with the dATP(N7) through T-shaped π–π (not indicated here), cation−π (pink dashed line), and hydrogen-bonding (black dashed line) interactions. (d) The amine side chain of K61 interacts with the dATP(N7) through cation−π (pink dashed line) and hydrogen-bonding (black dashed line) interactions. This side chain configuration of K61 also allows the formation of charge–charge and hydrogen-bonding interactions with one of the α-phosphate oxygens (black dashed line). (e) The amine side chain of K61 interacts with the dATP base through cation−π interactions (pink dashed line). This side chain configuration of K61 also allows hydrogen-bonding interactions with the 5′T(O4) of TTD (black dashed line). (f) The A61 side chain opens up more room for incoming water and Na⁺ ions in the active side, which compensate for the absence of a charged and bulky protein side chain. The hydrogen-bonding interactions between the dATP(N7) and nearby water, as well as 5′T of TTD and water, are shown as black dashed lines. A Na⁺ ion comes into the active site occasionally and forms a cation−π interaction with the dATP(N7) (pink dashed line).

Figure 4

Hydrogen-bonding interactions between the side chain of Q38 and the 3′T(O2) of TTD and between the R61 side chain and the dATP(N7) are shown as black dashed lines. The WC base-pairing interactions between the 3′T of TTD and dATP are also shown as black dashed lines.

Figure 5

Relative positioning of dATP, R61, and L62 in the S62L systems with a TTD (a) or a TT (b) motif. The WT R61 is shown in gray shades, and the mutant L62 is shown in purple shades along with their matching-colored labels. For both R61 and L62, one configuration is depicted at atomic level in gray and purple, respectively, and the other configurations are depicted as sticks that are oriented along the side chain. In the presence of a TTD, the R61 side chain adopts three configurations, while the L62 side chain adopts only two. In the presence of a TT, both the R61 and L62 side chains each exhibit one less configuration compared to the system with a TTD. The extending 5′T of the TT is unable to interact effectively with the side chains of R61 and L62, resulting in the elimination of some configurations found in the presence of the TTD.