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, singleturnover 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.

Mechanisms of Base Selection by Human Single-stranded Selective Monofunctional Uracil-DNA Glycosylase

“…Catalytic rate constants are presented in Table 1. Previous studies have established that E. coli MUG and hSMUG1 bind tightly to the abasic site-containing oligonucleotide following the removal of a target base (13,14,52). Due to the strong product binding displayed by some glycosylases, steady-state kinetic analysis does not give an accurate reflection of relative rates with different potential substrates.…”

“…2 of Ref. 52 for structures of modified purines paired with uracil). Duplex stability was determined by measuring the temperature dependence of the oligonucleotide UV absorbance at 260 nm in aqueous solution as a function of oligonucleotide concentration (Table 1).…”

“…In our previous study of a related glycosylase, E. coli MUG, three parameters were identified that are used by MUG to select a target pyrimidine (52). These factors were 1) duplex stability (pyrimidines in less stable duplex structures were repaired faster), 2) 5-substituent size (pyrimidine analogs with smaller 5-substituents were repaired faster than those with larger substituents), and 3) 5-substituent electronic inductive effects (pyrimidines with electron-withdrawing substituents in the 5-position were repaired faster than those with electrondonating substituents).…”

“…In a previous study, we demonstrated that the rate of base cleavage by MUG was inversely proportional to the size of the 5-substituent (52). In that study, we examined only spherical substituents whose size could be estimated from van der Waals radii and bond lengths.…”

Identification of Escherichia coli Mismatch-specific Uracil DNA Glycosylase as a Robust Xanthine DNA Glycosylase

“…2-3). It is known that mismatched uracilcontaining base pairs such as G/U are thermodynamically less stable than the A/U base pair (41). However, data on the stability of xanthine-containing base pairs are limited (42).…”