Characterization of the Escherichia coli uracil-DNA glycosylase.inhibitor protein complex.
The Bacillus subtilis bacteriophage PBS2 uracil-DNA glycosylase inhibitor (Ugi) protein was characterized and shown to form a stable complex with Escherichia coli uracil-DNA glycosylase (Ung). As determined by mass spectrometry, the Ugi protein had a molecular weight of 9,474. We confirmed this value by sedimentation equilibrium centrifugation and determined that Ugi exists as a monomeric protein in solution. Amino acid analysis performed on both Ugi and Ung proteins was in excellent agreement with the amino acid composition predicted from the respective nucleotide sequence of each gene. The Ung.Ugi complex was resolved from its constitutive components by nondenaturing polyacrylamide gel electrophoresis and shown to possess a 1:1 stoichiometry. Analytical ultracentrifugation studies revealed that the Ung.Ugi complex had a molecular weight of 35,400, consistent with the complex containing one molecule each of Ung and Ugi. The acidic isoelectric points of the protein species were 6.6 (Ung) and 4.2 (Ugi), whereas the Ung.Ugi complex had an isoelectric point of 4.9. Dissociation of the Ung.Ugi complex by SDS-polyacrylamide gel electrophoresis revealed no apparent alteration in the molecular weight of either polypeptide subsequent to binding. Furthermore, when the Ung.Ugi complex was treated with urea and resolved by urea-polyacrylamide gel electrophoresis, both uracil-DNA glycosylase and inhibitor activities were recovered from the dissociated complex. Thus, the complex seems to be reversible. In addition, we demonstrated that the Ugi interaction with Ung prevents enzyme binding to DNA and dissociates uracil-DNA glycosylase from a preformed DNA complex.
Bennett
S E
SE
Department of Agricultural Chemistry, Oregon State University, Corvallis 97331.
Mosbaugh
D W
DW
D000596:Amino Acids / Q000032:analysis; D017103:Bacillus Phages / Q000737:chemistry; D001426:Bacterial Proteins / Q000378:metabolism; D045647:DNA Glycosylases; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism*; D007526:Isoelectric Point; D046911:Macromolecular Substances; D008970:Molecular Weight; D009699:N-Glycosyl Hydrolases / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D051981:Uracil-DNA Glycosidase; D014764:Viral Proteins / Q000378:metabolism*
D000596:Amino Acids; D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D014764:Viral Proteins; C027619:uracil-DNA glycosylase inhibitor protein, B. subtilis bacteriophage; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase