Modulation of the turnover of formamidopyrimidine DNA glycosylase.

Prokaryotic and eukaryotic cells utilize a number of different mechanisms to repair constantly accumulating DNA damage due to environmental and endogenous chemical agents. In the absence of repair, many DNA lesions may block replication and transcription, while others may decrease replication fidelity. These disruptions may result in mutations and ultimately carcinogenesis in eukaryotes. The base excision repair (BER)1 pathway is a key component in the cellular response to DNA lesions (1). Formamidopyrimidine DNA glycosylase (Fpg) functions in the E. coli BER pathway as an N-glycosylase and abasic site (AP) lyase, with the predominant DNA incision product resulting from a δ-elimination reaction (2). The Fpg glycosylase activity results in the removal of formamidopyrimidine (Fapy) or 8-oxo-7,8-dihydroguanine (8-oxoG) lesions (3, 4). The catalytic mechanism of Fpg is similar to that of other BER glycosylases that manifest an AP lyase capability (5). The active site nucleophile is the N-terminal proline secondary amine (6). This nucleophile collapses onto C1′ of the scissile base deoxyribose moiety due to the developing electrophilic character at C1′, concomitant with the glycosylase step (5, Dodson, unpublished). The crystal and cocrystal structures of the E. coli Fpg and Fpg homologues from other bacterial and human sources have been published (7–11). Recent NMR analyses of free Fpg and Fpg bound to duplex DNA containing a site-specific AP site analogue, revealed a highly dynamic structure, even in complex with DNA (12). These data are consistent with molecular dynamics calculations of the enzyme in complex with 8-oxoG, even though the conclusions of these investigations differed concerning the anti/syn conformation of a bound 8-oxoG substrate (13–15). Precatalytic and catalytic mechanisms of Fpg have also been extensively investigated (13, 16–20). These investigations reveal a complex series of enzyme and DNA conformational changes that ultimately result in the formation of the Michaelis complex. Predictions concerning amino acid residues that could modulate precatalytic and catalytic steps based on structures, dynamics and spectroscopy have been successfully carried out, revealing molecular details of the overall catalytic mechanism (13). McCullough et al. (5) have discussed that the product spectra of the BER glycosylase-lyase enzymes arise as a consequence of kinetic competitions between sequential elimination events (glycosyl bond cleavage, β-elimination, δ-elimination) and hydrolysis of the corresponding covalent enzyme-product intermediates. Bhagwat and Gerlt (21) demonstrated that the Fpg δ-elimination step is dependent on a prior β-elimination step, a result that supports the kinetic competition argument. One corollary of that argument is that the cleavage of the covalent product from the enzyme is rate limiting in the catalytic scheme. The predominance of the δ-elimination product is consistent with the slow hydrolysis of Schiff base intermediates involving secondary amines. It has been suggested that the conserved Glu3 residue electrostatically stabilizes the electrophilic C1′ and subsequently, the protonated Schiff base intermediate (22, 23). Mutation of Lys57, whose e-amine contacts the phosphates one and two nucleotides 3′ to the 8-oxoG site, abolishes activity on 8-oxoG, but has little effect on AP lyase activity (8, 24). Gilboa et al. (8) have suggested that Lys57 protonates the leaving 3′ phosphate, thereby accelerating the β-elimination step. Results from studies of other combined glycosylase-AP lyases, such as T4 pyrimidine dimer glycosylase (T4-Pdg) and endonuclease VIII may also be used to suggest key amino acid residues involved in the catalytic mechanism of Fpg, due to a commonality in catalytic mechanisms. Meador et al. (25) showed that mutation of the His16 residue of T4-Pdg resulted in an enzyme that was severely compromised in turnover following the catalytic events. The His16 residue had been originally hypothesized to be involved in the formation of the Schiff base or subsequent intermediates. Further investigations did not support this hypothesis. The mutant enzymes were able to carry out the glycosylase step, form the Schiff base intermediate, and carry out the lyase step. However, the rate of product formation rapidly plateaued over time and was directly proportional to the amount of enzyme added. It was concluded that the His16 residue was involved in enzyme-product turnover. The mechanistic similarities between T4-Pdg and Fpg suggested that some residue in the Fpg structure might play a similar role to that of T4-Pdg His16. This study tests that hypothesis by identifying a similar candidate histidine residue, mutating that residue and investigating the alteration in catalytic properties of the mutant enzymes. The commonality of mutant enzyme phenotypes exists despite a complete lack of structural or sequence homology and entirely different active site architectures. The implication is that these residues catalyze the rate limiting and essential step for these BER DNA lyases.