Computational Study of the Force Dependence of Phosphoryl Transfer during DNA Synthesis by a High Fidelity Polymerase

High fidelity polymerases are efficient catalysts of phosphodiester bond formation during DNA replication or repair. We interpret molecular dynamics simulations of a polymerase bound to its substrate DNA and incoming nucleotide using a quasiharmonic model to study the effect of external forces applied to the bound DNA on the kinetics of phosphoryl transfer. The origin of the force dependence is shown to be an intriguing coupling between slow, delocalized polymerase-DNA modes and fast catalytic site motions. Using noncognate DNA substrates we show that the force dependence is context specific.

Figure 1

(Right) Solvated BF pol/DNA/dNTP ternary complex. (Left) Catalytic site showing key elements of the phosphoryl transfer reaction. A covalent bond is formed between $\alpha$-phosphorous of the incoming nucleotide and the $O{3}^{\prime }$ oxygen of the terminal primer base.

Figure 2

The potential surface along the catalytic reactive distance $X$ is given by $V_0=0.5k_x[X-X_A(0){]}^2$. An external force $\mathbf{F}$ shifts the equilibrium position of the ground state $A$ from $X_A(0)$ to $X_A(\mathbf{F})$ and that of the transition state $B$ from $X_B(0)$ to $X_B(\mathbf{F})$ to alter the barrier for catalysis. The new potential surface for $X$ is $V_{\mathbf{F}}=0.5k_x[X-X_A(\mathbf{F}){]}^2-[F_x/(2k_x)]$, where $F_x$ is the force alone $X$.

Figure 3

Correlations between vector displacements ($\mathbf{r}-⟨\mathbf{r}⟩$) of atoms for the $\mathbf{G}:\mathbf{C}$ and $\mathbf{G}:\mathbf{A}$ systems; vector $\mathbf{r}$ is from origin to the atom of interest, and $⟨\mathbf{r}⟩$ its average. Enlarged views for all 4 systems are provided in Figs. S8–S11 in 14.

Figure 4

Relative catalytic rate vs force on the DNA template strand. TSS: Transition state shift.