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Volume 271, Number 46, Issue of November 15, 1996 pp. 29170-29181
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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

(Received for publication, June 24, 1996, and in revised form, August 20, 1996)

Russell J. Sanderson ^§ and Dale W. Mosbaugh ^§¶

From the  Department of Agricultural Chemistry, the ^§ Department of Biochemistry and Biophysics, and the ^¶ Environmental Health Science Center, Oregon State University, Corvallis, Oregon 97331

The bacteriophage PBS2 uracil-DNA glycosylase inhibitor (Ugi) protein inactivates uracil-DNA glycosylase (Ung) by forming an exceptionally stable protein-protein complex in which Ugi mimics electronegative and structural features of duplex DNA (Beger, R. D., Balasubramanian, S., Bennett, S. E., Mosbaugh, D. W., and Bolton, P. H. (1995) J. Biol. Chem. 270, 16840-16847; Mol, C. D., Arvai, A. S., Sanderson, R. J., Slupphaug, G., Kavli, B., Krokan, H. E., Mosbaugh, D. W., and Tainer, J. A. (1995) Cell 82, 701-708). The role of specific carboxylic amino acid residues in forming the Ung·Ugi 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 Ung·Ugi 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 Ung·Ugi 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.

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