Structural Mechanism for the Fidelity Modulation of DNA Polymerase λ

Abstract

The mechanism of DNA polymerase (pol) fidelity is of fundamental importance in chemistry and biology. While high-fidelity pols have been well studied, much less is known about how some pols achieve medium or low fidelity with functional importance. Here we examine how human DNA polymerase λ (Pol λ) achieves medium fidelity by determining 12 crystal structures and performing pre-steady-state kinetic analyses. We showed that apo-Pol λ exists in the closed conformation, unprecedentedly with a preformed MgdNTP binding pocket, and binds MgdNTP readily in the active conformation in the absence of DNA. Since prebinding of MgdNTP could lead to very low fidelity as shown previously, it is attenuated in Pol λ by a hydrophobic core including Leu431, Ile492, and the Tyr505/Phe506 motif. We then predicted and demonstrated that L431A mutation enhances MgdNTP prebinding and lowers the fidelity. We also hypothesized that the MgdNTP-prebinding ability could stabilize a mismatched ternary complex and destabilize a matched ternary complex, and provided evidence with structures in both forms. Our results demonstrate that, while high-fidelity pols follow a common paradigm, Pol λ has developed specific conformations and mechanisms for its medium fidelity. Structural comparison with other pols also suggests that different pols likely utilize different conformational changes and microscopic mechanisms to achieve their catalytic functions with varying fidelities.

Uniform Free-Energy Profiles of the P–O Bond Formation and Cleavage Reactions Catalyzed by DNA Polymerases β and λ

“…When the closed, “catalytically competent”, ternary complexes of DNA polymerases are used for the simulations, the fidelity control originates from the preceding steps: thermodynamics of dNTP binding ,− or the deprotonation kinetics of the 3′-OH group of the primer DNA strand because these steps are more sensitive to “small” structural variations in the ternary complexes. When the enzyme carries out the reaction while in the (partially-)­open, “catalytically incompetent”, conformation, , the catalysis of the P–O bond formation/breakage reactions is greatly diminished. , …”

“…In search for the main source of the catalytic effect of Polβ, tPolλ, and tPolλΔL1, we explored the representative structures of the RS, IS, TS, and PS and asked what interactions are responsible for the higher stability of the TS in the enzyme compared to that in the solution. The characteristic feature of the active sites of Polβ, tPolλ, and tPolλΔL1 is a network of favorable electrostatic (including hydrogen-bonding) interactions, which interconnects the three apparent binding sites for the nucleobase, dRib, and triphosphate groups of dNTP. ,,, These interactions, present already in the RS and preserved during the entire nucleotidyl-transfer reaction, involve the sidechains of Arg149/386/386, Arg183/420/420, and Asn279/513/508, the backbone and sidechain of Tyr271/505/500, and the nucleobase of the templating nucleotide in Polβ/tPolλ/tPolλΔL1. In the solution reaction, the role of the amino acid residues and the templating nucleotide is fulfilled only suboptimally by water molecules, which are incapable of establishing an electrostatically preorganized reaction cage. ,, …”

Nonhomologous DNA end-joining for repair of DNA double-strand breaks

“…There has been remarkable progress in determining progressively higher resolution structures of some of the NHEJ proteins or, at least, portions of them (48,49,64,84,94,(107)(108)(109)(110)(111)(112) (Fig. 4).…”

Thermococcus sp. 9°N DNA polymerase exhibits 3′-esterase activity that can be harnessed for DNA sequencing

“…The oligonucleotides used in this work are listed in Supplementary Table 1. We first used pre-steady-state kinetic assays 14,15 to show that the substrates 3′-NL can be incorporated into DNA by 9°N-I in a single turnover event in the presence of Mg 2+ , though, as expected for substrate analogs, the k pol values of 3′-NL were lower than those of dNTP by a factor of 6–15, while the K d,app values were higher by a factor of 10–65 (Table 1).

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“…The reaction was quenched with 0.1 M EDTA, pH 8.0. A rapid quench instrument (KinTek Instrument Corp., State College, PA) was used for the reaction time ranging from 100 ms to 60 s at varying concentrations of nucleotides 14,15 . DNA substrates used in the extension assays are listed in Supplementary Table 1.…”