Role of His-16 in Turnover of T4 Pyrimidine Dimer Glycosylase

Abstract

Previously, the histidine residue at position 16 in the mature T4 pyrimidine dimer glycosylase (T4-PDG) protein has been suggested to be involved in general (nontarget) DNA binding. This interpretation is likely correct, but, in and of itself, cannot account for the most dramatic phenotype of mutants at this position: their inability to restore ultraviolet light resistance to a DNA repair-deficient Escherichia coli strain. Accordingly, this residue has been mutated to serine, glutamic, aspartic acid, lysine, cysteine, and alanine. The mutant proteins were expressed, purified, and their abilities to carry out several functions of T4-PDG were assessed. The mutant proteins were able to perform most functions tested in vitro, albeit at reduced rates compared with the wild type protein. The most likely explanation for the biochemical phenotypes of the mutants is that the histidine residue is required for rapid turnover of the enzyme. This role is interpreted and discussed in the context of a reaction mechanism able to account for the complete spectrum of products generated by T4-PDG during a single turnover cycle. The base excision repair (BER)1 glycosylases initiate DNA repair by recognizing inappropriate or damaged DNA bases and removing them via glycosyl bond scission (1). These enzymes are responsible for the substrate specificity of their particular BER pathways, a formidable accomplishment, as this recognition must proceed within the context of a vast excess of perfectly normal and appropriate bases. A subset of these enzymes also carry out scission of the DNA sugar-phosphate backbone by ␤-elimination and adjacent to the site of glycosyl bond cleavage. These enzymes will be referred to as "BER glycosylase-lyases." Enzymes in this subset of glycosylases utilize a primary or secondary amino group as an active site nucleophile. The amino group adds to C 1 Ј because of the developing electrophilic character of that carbon accompanying glycosyl bond cleavage. The resulting Schiff base enzyme-DNA intermediate, in its protonated form, has been proposed to kinetically assist DNA backbone cleavage (2). Gerlt (3) has hypothesized that the rate of the ␤-elimination reaction is directly determined by the acidity of the 2Ј-hydrogens on the sugar undergoing 3Ј-phosphate group elimination. Within this hypothesis, the role of the protonated Schiff base would be to lower the pK a of the 2Ј-hydrogens. In the proposed reaction scheme, one of these hydrogens would be abstracted by a general base, either an activated water or a basic amino acid side chain. T4-PDG is a ultraviolet light (UV) pyrimidine photodimer-specific BER glycosylase-lyase similar mechanistically to other BER glycosylase-lyases. The N-terminal threonine ␣-amino group is the active site nucleophile (4).To investigate those amino acid candidates whose side chains might serve as general bases for abstracting a sugar 2Ј-hydrogen to initiate the ␤-elimination reaction, we identified all amino acid side chains within 10 Å of the ␣-amino nitrogen in the cocrystal struc...

Toward a Detailed Understanding of Base Excision Repair Enzymes: Transition State and Mechanistic Analyses of N-Glycoside Hydrolysis and N-Glycoside Transfer

“…Mutational analysis of this cluster indicated a role in substrate binding rather than catalysis . Detailed analysis of His16 suggested a catalytic role in the lyase step . The T4PDG_Glu23Gln·(T◊T) structure 525 showed that Glu23 and O4‘ of the 3‘−T residue of the T◊T dimer were positioned within 4 Å of C1‘ of the target nucleotide and could be important in stabilizing an oxacarbenium ion or a Schiff base intermediate.…”

“…Similar structures effectively inhibited eMutY, hTDG, eMUG, and eUDG. 279,344,520,521 A number of catalytic mechanisms for T4PDG have been proposed based on trapping studies, 475,522 mutational analysis, 513,516,518,523,524 oligonucleotide analogues, 519 X-ray crystallography, 510,511,524,525 and computational simulations 491 (Figure 37). The T4PDG_Glu23Gln‚ (T)T) crystal structure 525 showed T4PDG_Gln23 < 4 Å from O2 of T)T, the O′4 ring oxygen, and NR of T4PDG_Thr2, suggesting several possible mechanistic roles.…”

“…516 Detailed analysis of His16 suggested a catalytic role in the lyase step. 523 The T4PDG_Glu23Gln‚(T)T) structure 525 showed that Glu23 and O4′ of the 3′-T residue of the T)T dimer were positioned within 4 Å of C1′ of the target nucleotide and could be important in stabilizing an oxacarbenium ion or a Schiff base intermediate. T4PDG inhibition studies gave K d ) 9 nM for a reduced AP site (RAP) compared with 17 nM for the 4-aza-dR mimic and 140 nM for THF.…”

Modulation of the Turnover of Formamidopyrimidine DNA Glycosylase

“…Results from studies of other combined glycosylase:APlyases, 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.…”

“…These reactions were incubated at 37 °C for 30 min, an equal amount of formamide loading buffer was added, and the samples analyzed as previously described. When carried out with an excess of DNA substrate, the fraction of DNA molecules forming a covalent intermediate, as measured by the gel migration pattern characteristic of covalent protein-DNA complexes (Meador, et al,25), should directly measure the active molecules in the enzyme preparation. Burst kinetics methods for determining the fraction of active enzyme molecules would be problematic since the apparent defect in the H71A mutant was in the turnover step.…”