Reversible Chemical Step and Rate-Limiting Enzyme Regeneration in the Reaction Catalyzed by Formamidopyrimidine-DNA Glycosylase

Abstract

Formamidopyrimidine-DNA N-glycosylase (Fpg) operates in the base excision repair pathway in bacteria by removing oxidized guanine bases from DNA and can also cleave the nascent or preformed abasic DNA by beta,delta-elimination. In this work, we have used the quench-flow technique (i) to show that the kinetics of processing of 7,8-dihydro-8-oxoguanine and abasic site lesions by Fpg from Escherichia coli involves a burst phase and a stationary phase, (ii) to establish the reaction kinetic scheme, and (iii) to calculate the rate constants for the reaction steps. A comparison of the quench-flow results with the data from earlier stopped-flow kinetics with tryptophan and 2-aminopurine fluorescence detection reveals that the cleaved product formation is initially reversible; it is followed by conformational changes in the enzyme and DNA molecules that represent the postchemical irreversible rate-limiting steps. We have applied mass spectrometry with electrospray ionization to follow the appearance and disappearance of transient covalent intermediates between Fpg and the substrate DNA. The overall rate-limiting step of the enzymatic reaction seems to be the release of Fpg from its adduct with the 4-oxo-2-pentenal remnant of the deoxyribose moiety formed as a result of DNA strand cleavage by beta,delta-elmination.

New Environment-Sensitive Multichannel DNA Fluorescent Label for Investigation of the Protein-DNA Interactions

“…14B). Again aPu fluorescent label is not sensitive for binding by Nei whereas in our previous works related with hOGG1 and Fpg enzymes this fluorescent base was a very informative probe for the study of enzyme-DNA interactions [12]–[14], [41], [42]. The increase in the aPu fluorescence intensity at times >100 s can be the result of the transition to the states with poorer stacking and/or quenching abilities of the neighbor bases as a consequence of the strand cleavage and dissociation of the enzyme-product complex (Fig.…”

Thermodynamics of the DNA Damage Repair Steps of Human 8-Oxoguanine DNA Glycosylase

“…As it was recently demonstrated for another oxoG excision DNA glycosylase Fpg from E. coli , belonging to the Fpg/Nei family of DNA glycosylases, the determination of the thermodynamic parameters of the substrate binding and lesion removal steps provided important information about oxoG recognition process [27], [29], [30]. The pre-steady-state kinetic data were obtained using pyrrolocytosine fluorescence in DNA [27].…”

Conformational Dynamics of DNA Repair by Escherichia coli Endonuclease III

“…8). As in the case of previously studied DNA glycosylases, E. coli Fpg (28,29,31,63) and human OGG1 (20,21), binding of Endo III to DNA begins most likely with an insertion of the wedge amino acids, Gln 41 and Leu 81 , into the helix (61,62). Insertion of other amino acids and eversion of the damaged nucleotide into the active site proceed at the next stage of specific lesion recognition, resulting in "stapling" of the enzyme on DNA.…”

“…Stopped-flow Experiments-The experiments were conducted essentially as described previously (27)(28)(29). All experiments were carried out at 25°C in buffer containing 50 mM Tris-HCl (pH 7.5), 50 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, and 7% glycerol (v/v).…”