Abstract
The fidelity of DNA polymerases (Pols) refers to their ability to incorporate the correct nucleotide into the growing strand during DNA synthesis. Each Pol operates with a certain degree of fidelity, from high (∼10–8; ∼1 error in 108 bases) to low (∼10–1; ∼1 error in 10 bases) values. The mechanistic factors behind these differences in fidelity are poorly understood. Here, we show that the formation of the Michaelis–Menten complex is critically affected by the metal-mediated dynamics of local structural features at the catalytic center of Pols. We demonstrated this by integrating recent structural and kinetics data of high-fidelity Pol β and low-fidelity Pol η with equilibrium molecular dynamics and free-energy simulations of paired and mispaired reactant complexes of these Pols. We found that local dynamics at the reaction center determines whether the nucleophile is optimally aligned to incorporate the correct (dCTP) or incorrect (dATP) nucleotide opposite a template deoxyguanosine (dG). In Pol β, local structural distortions at the catalytic site are visible only in the dG:dATP mispair complex, which energetically disfavors incorrect nucleotide addition and thus promotes high fidelity. In contrast, in Pol η we observed a more flexible base pair shape complementarity at the catalytic site. This allows reactive configurations of matched and mismatched complexes to be formed with similar ease, thus explaining the low fidelity of Pol η in line with the experimental evidence. Comparisons with other Pols suggest that these local metal-mediated structural dynamics at the reaction center of the catalytic site are crucial to modulating Pol fidelity.
Highlights
The fidelity of DNA polymerase (Pol) enzymes refers to their ability to precisely duplicate the original template during DNA synthesis,[1−3] with implications in biology, drug discovery, and biotechnology.[4−10] Mechanistically, high-fidelity Pols efficiently select the correct deoxyribonucleoside triphosphate from solution and add it to the 3′-end of the growing primer strand, forming a canonical Watson−Crick (WC) base pair
The chemical step for incorporating the incoming deoxyribonucleoside triphosphate (dNTP) to the terminal primer is often proposed as the rate-limiting step, while reactant positioning and alignment at the catalytic site are thought to be critical for prompt nucleotide incorporation.[1,13−19] During this step, dNTPs are selected and recruited from solution to form a ternary dNTP−DNA−Pol complex, leading to dNTP addition
The mismatched Pol β complex was modeled on PDB ID 4LVS,[24] where the dG:dATP mispair is in the anti−anti conformation that is distorted compared to the anti−syn conformation commonly observed in isolated DNA duplexes.[49,50]
Summary
The fidelity of DNA polymerase (Pol) enzymes refers to their ability to precisely duplicate the original template during DNA synthesis,[1−3] with implications in biology, drug discovery, and biotechnology.[4−10] Mechanistically, high-fidelity Pols efficiently select the correct deoxyribonucleoside triphosphate (dNTP) from solution and add it to the 3′-end of the growing primer strand, forming a canonical Watson−Crick (WC) base pair. The chemical step for incorporating the incoming dNTP to the terminal primer is often proposed as the rate-limiting step, while reactant positioning and alignment at the catalytic site are thought to be critical for prompt nucleotide incorporation (see below).[1,13−19] During this step, dNTPs are selected and recruited from solution to form a ternary dNTP−DNA−Pol complex, leading to dNTP addition This involves the nucleophilic attack of the deprotonated 3′-OH group of the terminal primer residue [P(−1)] on the Pα atom of the incoming dNTP via an in-line SN2-like mechanism (Scheme 1).[20,21] The release of the pyrophosphate (PPi) leaving group occurs each time dNTP is incorporated and is likely facilitated by an additional transient third metal ion.[19,22−28] The overall Pol-mediated DNA duplication process often involves a large conformational change of the Pol enzyme from an open structure to a closed structure. The nucleotide-binding or fingers domain of a Pol moves inward and covers the catalytic domain (Figure 1).[29−31] This substrate-induced conformational change is believed to be necessary to achieve the proper placement of the right dNTP within the two-metal catalytic site of Pols.[32−34]
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