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, 25 (6), 2169-76

Mechanism of Efficient and Accurate Nucleotide Incorporation Opposite 7,8-dihydro-8-oxoguanine by Saccharomyces Cerevisiae DNA Polymerase Eta

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Mechanism of Efficient and Accurate Nucleotide Incorporation Opposite 7,8-dihydro-8-oxoguanine by Saccharomyces Cerevisiae DNA Polymerase Eta

Karissa D Carlson et al. Mol Cell Biol.

Abstract

Most DNA polymerases incorporate nucleotides opposite template 7,8-dihydro-8-oxoguanine (8-oxoG) lesions with reduced efficiency and accuracy. DNA polymerase (Pol) eta, which catalyzes the error-free replication of template thymine-thymine (TT) dimers, has the unique ability to accurately and efficiently incorporate nucleotides opposite 8-oxoG templates. Here we have used pre-steady-state kinetics to examine the mechanisms of correct and incorrect nucleotide incorporation opposite G and 8-oxoG by Saccharomyces cerevisiae Pol eta. We found that Pol eta binds the incoming correct dCTP opposite both G and 8-oxoG with similar affinities, and it incorporates the correct nucleotide bound opposite both G and 8-oxoG with similar rates. While Pol eta incorporates an incorrect A opposite 8-oxoG with lower efficiency than it incorporates a correct C, it does incorporate A more efficiently opposite 8-oxoG than opposite G. This is mainly due to greater binding affinity for the incorrect incoming dATP opposite 8-oxoG. Overall, these results show that Pol eta replicates through 8-oxoG without any barriers introduced by the presence of the lesion.

Figures

FIG. 1.
FIG. 1.
Structures of the 8-oxoG · C and 8-oxoG · A base pairs. The anti and syn designations refer to the relative orientation of the base and sugar around the glycosidic bond.
FIG. 2.
FIG. 2.
Biphasic kinetics of C incorporation opposite G and an 8-oxoG. (A) Preincubated Pol η (33 nM) and the template G DNA substrate (100 nM) were mixed with dCTP (20 μM) by using a rapid chemical quench flow instrument for various reaction times. The amounts of product formed (•) were graphed as a function of time, and the data were fit to the burst equation with an amplitude equal to 25 ± 3 nM and a rate constant for the exponential phase equal to 1.5 ± 0.5 s−1. (B) Preincubated Pol η (33 nM) and the template 8-oxoG DNA substrate (100 nM) were mixed with dCTP (20 μM) for various reaction times. The data were fit to the burst equation with an amplitude equal to 20 ± 1 nM and a rate constant for the exponential phase equal to 1.4 ± 0.2 s−1.
FIG. 3.
FIG. 3.
Active-site titration and Kd for DNA binding. (A) Preincubated Pol η (33 nM) and various concentrations of template G DNA (•, 10 nM; ○, 20 nM; ▪, 30 nM; □, 50 nM; ▴, 75 nM; ▵, 100 nM) were mixed with dCTP (20 μM) for various reaction times. The solid lines represent the best fits to the burst equation. (B) Amplitudes of the exponential phases (•) were graphed as a function of total DNA concentration. The solid line represents the best fit to the quadratic equation, with an active-site concentration equal to 33 ± 1 nM and a Kd for the Pol η-DNA complex equal to 22 ± 2 nM. (C) Preincubated Pol η (33 nM) and various concentrations of template 8-oxoG DNA (•, 10 nM; ○, 20 nM; ▪, 30 nM; □, 50 nM; ▴, 75 nM; ▵, 100 nM) were mixed with dCTP (20 μM) for various reaction times. The solid lines represent the best fits to the burst equation. (D) Amplitudes of the exponential phases (•) were graphed as a function of total DNA concentration. The solid line represents the best fit to the quadratic equation, with an active-site concentration equal to 23 ± 1 nM and a Kd for the Pol η-DNA complex equal to 17 ± 3 nM.
FIG. 4.
FIG. 4.
Kinetics of dCTP incorporation opposite G and 8-oxoG. (A) Preincubated Pol η (33 nM) and the template G DNA substrate (100 nM) were mixed with various concentrations of dCTP (•, 0.5 μM; ○, 1 μM; ▪, 2 μM; □, 5 μM; ▴, 10 μM; ▴, 20 μM) for various reaction times. The solid lines represent the best fits to the burst equation. (B) Observed rate constants of the exponential phases (•) were graphed as a function of dCTP concentration. The solid line represents the best fit to the hyperbolic equation, with a kpol equal to 2.1 ± 0.2 s−1 and a Kd for the Pol η-DNA-dCTP complex equal to 4.5 ± 1.2 μM. (C) Preincubated Pol η (33 nM) and template 8-oxoG DNA substrate (100 nM) were mixed with various concentrations of dCTP (•, 0.5 μM; ○, 1 μM; ▪, 2 μM; □, 5 μM; ▴, 10 μM; ▵, 20 μM) for various reaction times. The solid lines represent the best fits to the burst equation. (D) Observed rate constants of the burst phases (•) were graphed as a function of dCTP concentration. The solid line represents the best fit to the hyperbolic equation, with a kpol equal to 2.2 ± 0.5 s−1 and a Kd for the Pol η-DNA-dCTP complex equal to 9.4 ± 4.8 μM.
FIG. 5.
FIG. 5.
Kinetics of dATP incorporation opposite G and 8-oxoG. (A) Preincubated Pol η (33 nM) and the template G DNA substrate (100 nM) were mixed with various concentrations of dATP (•, 50 μM; ○, 100 μM; ▪, 200 μM; □, 500 μM; ▴, 1,000 μM; ▵, 2,000 μM) for various reaction times. The solid lines represent the best fits to the linear equation. (B) Observed rate constants (•) were graphed as a function of dATP concentration, and the solid line represents the best fit to the hyperbolic equation, with a kpol of 0.012 ± 0.001 s−1 and a Kd for the Pol η-DNA-dATP complex equal to 120 ± 50 μM. (C) Preincubated Pol η (33 nM) and the template 8-oxoG DNA substrate (100 nM) were mixed with various concentrations of dATP (•, 2 μM; ○, 5 μM; ▪, 10 μM; □, 20 μM; ▴, 50 μM; ▵, 100 μM) for various reaction times. The solid lines represent the best fits to the linear equation. (D) Observed rate constants (•) were graphed as a function of dATP concentration. The solid line represents the best fit to the hyperbolic equation, with a kpol of 0.022 ± 0.001 s−1 and a Kd for the Pol η-DNA-dATP complex equal to 6.5 ± 1.2 μM.
FIG. 6.
FIG. 6.
Free energy diagrams of a portion of the nucleotide incorporation reaction. The left panel represents the incorporation of C (black) and A (gray) opposite template G, and the right panel represents the incorporation of C and A opposite template 8-oxoG. The ground state of the Pol η-DNA-dNTP complex is labeled E-DNA-dNTP, and the transition state of the nucleotide incorporation step is labeled (E-DNA-dNTP). To calculate these values, we have assumed arbitrary concentrations of DNA equal to 100 nM and dNTP equal to 100 μM.

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