Mechanism of nucleotide incorporation opposite a thymine-thymine dimer by yeast DNA polymerase eta - PubMed (original) (raw)
Mechanism of nucleotide incorporation opposite a thymine-thymine dimer by yeast DNA polymerase eta
M Todd Washington et al. Proc Natl Acad Sci U S A. 2003.
Abstract
DNA polymerase eta (Poleta) has the unique ability to replicate through UV-light-induced cyclobutane pyrimidine dimers. Here we use pre-steady-state kinetic analyses to examine the mechanism of nucleotide incorporation opposite a cis-syn thymine-thymine (TT) dimer and an identical nondamaged sequence by yeast Poleta. Poleta displayed "burst" kinetics for nucleotide incorporation opposite both the damaged and nondamaged templates. Although a slight decrease occurred in the affinity (Kd) of nucleotide binding opposite the TT dimer relative to the nondamaged template, the rate (kpol) of nucleotide incorporation was the same whether the template was damaged or nondamaged. These results strongly support a mechanism in which the nucleotide is directly inserted opposite the TT dimer by using its intrinsic base-pairing ability without any hindrance from the distorted geometry of the lesion.
Figures
Fig. 1.
Pre-steady-state kinetics of nucleotide incorporation opposite a TT dimer (indicated by ν) and the analogous nondamaged sequence (both shown above the graphs) by Polη and active-site titrations. (A) Polη (120 nM) and the damaged 3′ T DNA substrate (300 nM) were mixed with dATP (500 μM) by using a rapid chemical quench flow instrument for various reaction times. The data were fit to the burst equation with an amplitude of 100 ± 3 nM and rate constants equal to 1.0 ± 0.06 s–1 and 0.050 ± 0.003 s–1. (B) Polη (120 nM) and various concentrations of the damaged 3′ T DNA substrate (•, 25 nM; ○, 50 nM; ▪, 100 nM; □, 150 nM; ▴, 200 nM; ▵, 300 nM) were mixed with dATP (500 μM) for various reaction times. The solid lines represent the best fits to the burst equation. (C) The amplitudes of the burst phases in B (•) were graphed as a function of DNA concentration, and the solid line represents the best fit to the quadratic equation with a for the Polη–DNA complex equal to 40 ± 5 nM and an active-site concentration equal to 120 ± 4 nM. (D) Polη (120 nM) and the nondamaged 3′ T DNA substrate (300 nM) were mixed with dATP (200μM) for various reactions times. The data were fit to the burst equation with an amplitude of 100 ± 2 nM and rate constants equal to 1.4 ± 0.06 s–1 and 0.054 ± 0.002 s–1. (E) Polη (120 nM) and various concentrations of the nondamaged 3′ T DNA substrate (•, 25nM; ○, 50nM; ▪, 100 nM; □, 150 nM; ▴, 200 nM; ▵, 300 nM,) were mixed with dATP (300 μM) for various reaction times. The solid lines represent the best fits to the burst equation. (F) The amplitudes of the burst phases in E (•) were graphed as a function of DNA concentration, and the solid line represents the best fit to the quadratic equation with a for the Polη–DNA complex equal to 32 ± 2 nM and an active-site concentration equal to 120 ± 2 nM.
Fig. 2.
Nucleotide incorporation opposite the 3′ T of the TT dimer and the analogous nondamaged template residue. (A) Polη (120 nM) and the damaged 3′ T DNA substrate (300 nM) were mixed with various concentrations of dATP (•, 10 μM; ○, 20 μM; ▪, 50 μM; □, 100 μM; ▴, 200 μM; ▵, 500 μM) for various reaction times. The solid lines represent the best fits to the burst equation. (B) The observed rate constants of the burst phases in A (•) were graphed as a function of dATP concentration, and the solid line represents the best fit to the hyperbolic equation with a _k_pol equal to 1.2 ± 0.07 s–1 and a for the Polη–DNA–dATP complex equal to 130 ± 20 μM. (C) Polη (120 nM) and the nondamaged 3′ T DNA substrate (300 nM) were mixed with various concentrations of dATP (•, 5 μM; ○, 10 μM; ▪, 20 μM; □, 50 μM; ▴, 100 μM; ▵, 200 μM) for various reaction times. The solid lines represent the best fits to the burst equation. (D) The observed rate constants of the burst phases in C (•) were graphed as a function of dATP concentration, and the solid line represents the best fit to the hyperbolic equation with a _k_pol equal to 1.5 ± 0.08 s–1 and a for the Polη–DNA–dATP complex equal to 28 ± 5 μM.
Fig. 3.
Nucleotide incorporation opposite the 5′ T of the TT dimer and the analogous nondamaged template residue. (A) Polη (120 nM) and the damaged 5′ T DNA substrate (300 nM) were mixed with various concentrations of dATP (•, 10 μM; ○, 20 μM; ▪, 50 μM; □, 100 μM; ▴, 200 μM; ▵, 500 μM) for various reaction times. The solid lines represent the best fits to the burst equation. (B) The observed rate constants of the burst phases in A (•) were graphed as a function of dATP concentration, and the solid line represents the best fit to the hyperbolic equation with a _k_pol equal to 2.5 ± 0.1 s–1 and a for the Polη–DNA–dATP complex equal to 72 ± 11 μM. (C) Polη (120 nM) and the nondamaged 5′ T DNA substrate (300 nM) were mixed with various concentrations of dATP (•, 5 μM; ○, 10 μM; ▪, 20 μM; □, 50 μM; ▴, 100 μM; ▵, 200 μM) for various reaction times. The solid lines represent the best fits to the burst equation. (D) The observed rate constants of the burst phases in C (•) were graphed as a function of dATP concentration, and the solid line represents the best fit to the hyperbolic equation with a _k_pol equal to 1.6 ± 0.04 s–1 and a for the Polη–DNA–dATP complex equal to 39 ± 3 μM.
Fig. 4.
Nucleotide incorporation opposite the abasic site (indicated by x in the sequence above the graphs). (A) Polη (120 nM) and the abasic site DNA substrate (300 nM) were mixed with various concentrations of dATP (•, 20 μM; ○, 50 μM; ▪, 100 μM; □, 200 μM; ▴, 500 μM; ▵, 1,000 μM) for various reaction times. The position of the abasic site is designated x in the given sequence. The solid lines represent the best fits to the linear equation. (B) The observed linear rate constants in A (•) were graphed as a function of dATP concentration, and the solid line represents the best fit to the hyperbolic equation with a _k_pol equal to 0.012 ± 0.001 s–1 and a for the Polη–DNA–dATP complex equal to 70 ± 10 μM.
Fig. 5.
Two possible models for the insertion of nucleotides opposite a TT dimer by Polη. In the “A rule” insertion model (Left), Polη flips both bases of the TT dimer out of its active site and inserts the first A opposite the resultant transient abasic site-like intermediate. Polη then flips the TT dimer inside its active site and directly inserts the second A opposite the 5′ T of the dimer. In the direct-insertion model (Right), Polη retains both bases of the TT dimer in its active site and directly inserts an A opposite both the 3′ T and 5′ T of the dimer.
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