Identification of Specific Carboxyl Groups on Uracil-DNA Glycosylase Inhibitor Protein That Are Required for Activity

Abstract

. (1995) Cell 82, 701-708). The role of specific carboxylic amino acid residues in forming the Ung⅐Ugi complex was investigated using selective chemical modification techniques. Ugi treated with carbodiimide and glycine ethyl ester produced five discrete protein species (forms I-V) that were purified and characterized. Analysis by mass spectrometry revealed that Ugi form I escaped protein modification, and forms II-V showed increasing incremental amounts of acyl-glycine ethyl ester adduction. Ugi forms II-V retained their ability to form a Ung⅐Ugi complex but exhibited a reduced ability to inactivate Escherichia coli Ung, directly reflecting the extent of modification. Competition experiments using modified forms II-V with unmodified Ugi as a competitor protein revealed that unmodified Ugi preferentially formed complex. Furthermore, unmodified Ugi and poly(U) were capable of displacing forms II-V from a preformed Ung⅐Ugi complex but were unable to displace Ugi form I. The primary sites of acyl-glycine ethyl ester adduction were located in the ␣2-helix of Ugi at Glu-28 and Glu-31. We infer that these two negatively charged amino acids play an important role in mediating a conformational change in Ugi that precipitates the essentially irreversible Ung/Ugi interaction.

Site-directed Mutagenesis and Characterization of Uracil-DNA Glycosylase Inhibitor Protein

“…in promoting stable Ung⅐Ugi complex formation (19). The unique ability of Ugi E28L to form a stable but reversible complex when challenged with wild type Ugi indicates that this residue plays a critical role in forming the locked complex.…”

“…Nonradioactive Ung (fraction V) was purified from E. coli JM105/pSB1051 grown in LB medium supplemented with 100 g/ml ampicillin (19 3 H]Ung in buffer A (30 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1 mM dithiothreitol, 5% (w/v) glycerol) containing 50 mM NaCl and incubated at 25°C for 10 min and then at 4°C for 20 min. Following complex formation, each sample was applied to a DE52 cellulose column equilibrated in buffer A containing 50 mM NaCl, washed with equilibration buffer, and step-eluted, as described previously (19). Fractions (1 ml) were collected and samples were analyzed for 3 H radioactivity.…”

“…Fifth, charge neutralization by carbodiimide-mediated adduction of Ugi carboxylic acid residues caused a decrease in inhibitor protein activity (19). Finally, chemical adduction of specific glutamic acid residues (Glu-28 and Glu-31) of Ugi located in the ␣2-helix correlated with the formation of an unstable Ung⅐Ugi complex (19).…”

“…Fourth, the recent x-ray structure of human uracil-DNA glycosylase complexed with a 10-bp oligonucleotide containing a target G⅐U mispair reveals the DNA complexed at the same site as Ugi (18). Fifth, charge neutralization by carbodiimide-mediated adduction of Ugi carboxylic acid residues caused a decrease in inhibitor protein activity (19). Finally, chemical adduction of specific glutamic acid residues (Glu-28 and Glu-31) of Ugi located in the ␣2-helix correlated with the formation of an unstable Ung⅐Ugi complex (19).…”

Escherichia coli nucleoside diphosphate kinase does not act as a uracil-processing DNA repair nuclease

“…To further examine the nature of the uracil-DNA glycosylase activity associated with the Ndk (Fraction III) preparation, inhibition studies were conducted with Ugi. The molecular mechanism of Ugi-mediated inhibition of Ung has been elucidated in detail by using both biochemical and structural approaches (18,26,28,30,31). By using the mechanism of DNA mimicry, Ugi specifically targets the conserved DNA-binding domain of Ung-family enzymes to form a tightly bound noncovalent Ung:Ugi complex with 1:1 stoichiometry (18,26).…”