Exploring the roles of nucleobase desolvation and shape complementarity during the misreplication of O(6)-methylguanine

Abstract

O(6)-methylguanine (O(6)-MeG) is a miscoding DNA lesion arising from the alkylation of guanine. This report uses the bacteriophage T4 DNA polymerase as a model to probe the roles of hydrogen-bonding interactions, shape/size, and nucleobase desolvation during the replication of this miscoding lesion. This was accomplished by using transient kinetic techniques to monitor the kinetic parameters for incorporating and extending natural and nonnatural nucleotides. In general, the efficiency of nucleotide incorporation does not depend on the hydrogen-bonding potential of the incoming nucleotide. Instead, nucleobase hydrophobicity and shape complementarity appear to be the preeminent factors controlling nucleotide incorporation. In addition, shape complementarity plays a large role in controlling the extension of various mispairs containing O(6)-MeG. This is evident as the rate constants for extension correlate with proper interglycosyl distances and symmetry between the base angles of the formed mispair. Base pairs not conforming to an acceptable geometry within the polymerase's active site are refractory to elongation and are processed via exonuclease proofreading. The collective data set encompassing nucleotide incorporation, extension, and excision is used to generate a model accounting for the mutagenic potential of O(6)-MeG observed in vivo. In addition, kinetic studies monitoring the incorporation and extension of nonnatural nucleotides identified an analog that displays high selectivity for incorporation opposite O(6)-MeG compared to unmodified purines. The unusual selectivity of this analog for replicating damaged DNA provides a novel biochemical tool to study translesion DNA synthesis.

Figure 1

(A) Structures for C:O⁶-MeG and T:O⁶-MeG based upon Watson-Crick hydrogen bonding capabilities. (B) DNA substrates used for all kinetic analyses. MeG denotes O⁶-methylguanine.

Figure 2

(A) Interplay of polymerization, extension, and exonuclease proofreading in the maintenance of genomic fidelity during DNA replication. (B) Minimal kinetic mechanism for DNA polymerization. Step 1 represents dNTP binding to the polymerase:nucleic acid complex. Step 2 represents the conformational change prior to phosphoryl transfer. Step 3 represents phosphoryl transfer. Step 4 represents the conformational change after phosphoryl transfer. Step 5 represents the release of the first product, pyrophosphate. Step 6 collectively represents translocation of the enzyme to the next insertion position and binding of the next correct dNTP. Abbreviations are as follows: Pol = bacteriophage T4 DNA polymerase, DNA_n= DNA substrate, Pol’ = conformational change in DNA polymer ase, PP_i = inorganic pyrophosphate, and DNA_n+1 = DNA product (DNA extended by one nucleobase).

Figure 3

(A) gp43 exo⁻ (1000 nM) and 5′-labeled 13/20MeG-mer (250 nM) were preincubated, mixed with 10 mM Mg²⁺ and variable concentrations of dCTP to initiate the reaction, and quenched with 500 mM EDTA at variable times (0.005–0.25 sec). The incorporation of dCTP was analyzed by denaturing gel electrophoresis. dCTP concentrations were 5 μM (O), 10 μM (●), 25 μM (➌), 50 μM (■), 100 μM (△), and 200 μM (▲). The solid lines represent the fit of the data to a single exponential. (B) The plot of observed rate constants for dCTP incorporation (O) versus dCTP concentration are hyperbolic. A fit of the data to the Michaelis-Menten equation was used to provide a k_pol of 1.5 +/− 0.2 sec⁻¹ and a K_d of 48+/− 16 μM.

Figure 4

(A) Structures of the three non-natural nucleotides used in this study (P-nucleotide, Zebularine, and 4-MePoTP). (B) Plot of the observed rate constant for incorporating P-nucleotide opposite O⁶-MeG (O) as a function of concentration of P-nucleotide concentration. Under the concentrations tested, the plot is linear and provides a value of 21,000 +/−8,000 M⁻¹sec⁻¹ for the catalytic efficiency for incorporating P-nucleotide opposite O⁶-MeG. (C) The plot of observed rate constants for 4-MePoTP incorporation (O) versus nucleotide concentration. A fit of the data to the Michaelis-Menten equation was used to provide a k_pol of 18 +/− 1.2 sec⁻¹ and a K_d of 202 +/− 30 μM for 4-MePoTP opposite O⁶-MeG.

Figure 5

(A) Experimental protocol used to monitor extension beyond correct or mismatched base pairs. See text for details. (B) Extension of natural and non-natural mispairs containing O⁶-MeG by gp43 exo⁻. Experiments were performed by adding a fixed concentration of natural (dCTP or dTTP) or non-natural nucleotide (P-nucleotide, Zebularine, or 4-MePoTP) to a preincubated solution of 1 μM gp43 exo^- and 500 nM 13/20MeG-mer. An aliquot of the reaction was quenched with 200 mM EDTA. After this time, 500 μM dGTP was added to initiate elongation beyond the mispair. An aliquot of the reaction was quenched with 200 mM EDTA after 1 minute (natural dNTPs) or 5 minutes (non-natural nucleotides). Reaction products are separated on a 20% denaturing polyacrylamide gel. The lane demarcated as “0” depicts unextended primer/template. All other lanes are labeled according to the nucleotides added. (C) Extension of various mispairs by gp exo⁺. All assays were performed using similar protocols described above using gp43 exo⁻.

Figure 6

(A) Kinetics of extension beyond a C:G base pair. 1 μM gp43 exo⁻ was preincubated with 500 nM 13/20G-mer, mixed with 10 mM Mg²⁺ and 25 μM dCTP/25 μM dGTP to initiate the reaction, and quenched with 500 mM EDTA at variable times (0.005–0.15 sec). Reaction products are separated on a 20% denaturing polyacrylamide gel. Lanes demarcated as “0” depict unextended primer/template only. (B) The data obtained from gel electrophoresis show the sequential accumulation of products. Product formation is denoted as follows: ○ = 14-mer, ● = 15-mer, ◇ = 16-mer, ■ = 17-mer, and ➌ = 18-mer. Fits of the data in product formation were generated using the rate constants denoted in Table 2 which were obtained through computer simulation of the data. (C) Time course for extension beyond a T:O⁶-MeG mismatch catalyzed by gp43 exo^-. Product formation is denoted as follows: ○ = 14-mer, ● = 15-mer, ➌ = 16-mer, ■ = 17-mer, △ = 18-mer, ▲ = 19-mer, and ◇ = 20-mer. The curves for product formation were generated using the rate constants denoted in Table 2 obtained through computer simulation (20) of the data.

Figure 7

Representation of the selectivity factor for natural and non-natural nucleotides for incorporation opposite O⁶-methylguanine. Selectivity factor is defined as (k_pol/K_d)_O6-MeG/(k_pol/K_d)_{G or A.} As illustrated, 4-MePoTP displays a selectivity factor of ~100 for incorporation opposite O⁶-methylguanine whereas other nucleotides tested show selectivity factors of less than 1.

Figure 8

Model for the coordination of polymerization and exonuclease activity during the misreplication of the miscoding DNA lesion, O⁶-methylguanine. See text for details.

Figure 9

Models for correct and translesion DNA synthesis involving the coordinated activities of replicative and specialized DNA polymerases. See text for details.