Mechanisms of base selection by the Escherichia coli mispaired uracil glycosylase

Abstract

The repair of the multitude of single-base lesions formed daily in cells of all living organisms is accomplished primarily by the base excision repair pathway that initiates repair through a series of lesion-selective glycosylases. In this article, single-turnover kinetics have been measured on a series of oligonucleotide substrates containing both uracil and purine analogs for the Escherichia coli mispaired uracil glycosylase (MUG). The relative rates of glycosylase cleavage have been correlated with the free energy of helix formation and with the size and electronic inductive properties of a series of uracil 5-substituents. Data are presented that MUG can exploit the reduced thermodynamic stability of mispairs to distinguish U:A from U:G pairs. Discrimination against the removal of thymine results primarily from the electron-donating property of the thymine 5-methyl substituent, whereas the size of the methyl group relative to a hydrogen atom is a secondary factor. A series of parameters have been obtained that allow prediction of relative MUG cleavage rates that correlate well with observed relative rates that vary over 5 orders of magnitude for the series of base analogs examined. We propose that these parameters may be common among DNA glycosylases; however, specific glycosylases may focus more or less on each of the parameters identified. The presence of a series of glycosylases that focus on different lesion properties, all coexisting within the same cell, would provide a robust and partially redundant repair system necessary for the maintenance of the genome.

FIGURE 1.

Sequences of the oligonucleotides examined for this study, where X = U, T, 5-fluorouracil, 5-chlorouracil, 5-bromouracil, or 5-iodouracil. In the purine series, P = A or G, hypoxanthine, purine, 2-aminoadenine, or 2-aminopurine. A, 24-mer oligonucleotide used for the enzyme kinetic experiments. B, 12-mer oligonucleotides used for the thermodynamic experiments.

FIGURE 2.

Proposed conformers of uracil base pairs with purine analogue base pairs in this study. R = H for uracil.

FIGURE 3.

Kinetic study of MUG cleavage of 5-substituted uracil analogues paired with guanine illustrating the gel electrophoretic assay (left) and time-dependent product ratio (right). Single turnover reactions were performed at 25 °C with 1.4 nm substrate and 0.56 μm MUG in the standard reaction buffer. A rapid quench-flow apparatus was used for reactions conducted from 53 ms to 200 s. Upper, FU:G as a substrate; lower, U:G as a substrate.

FIGURE 4.

Relationship between helix formation energy, ΔG, and glycosylase kinetics. The natural logarithm of the observed rate constant (ln k_obs) of the MUG cleavage reaction is plotted versus ΔG (kcal/mol).

FIGURE 5.

Relationship between substituent size and glycosylase cleavage rates. The observed rate constants (k_obs) for the MUG cleavage reactions are plotted against the size of the halogen substituents at the C(5) position of the substituted uracil paired with guanine.

FIGURE 6.

Relationship between electronic-inductive properties and glycosylase cleavage rates. The observed rate constants (k_obs) for the MUG cleavage reactions are plotted against the electronic inductive properties (σ_m) of the substituent at the C(5) position of the substituted uracil paired with guanine.

FIGURE 7.

Relationship between expected relative rates and observed relative rates based on the helix formation energy of the oligonucleotide, size, and electronic inductive property of the uracil C(5) substituent plotted on a log-log scale. The relative rates are calculated with respect to the rate of cleavage of the U:G base pair.