PMID:8063831
| Citation |
Bennett, SE, Jensen, ON, Barofsky, DF and Mosbaugh, DW (1994) UV-catalyzed cross-linking of Escherichia coli uracil-DNA glycosylase to DNA. Identification of amino acid residues in the single-stranded DNA binding site. J. Biol. Chem. 269:21870-9 |
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| Abstract |
Photochemical cross-linking of Escherichia coli uracil-DNA glycosylase (Ung) to oligonucleotide dT20 was performed to identify amino acid residues that reside in or near the DNA-binding site. UV-catalyzed cross-linking reactions produced a covalent Ung x dT20 complex which was resolved from uncross-linked enzyme by SDS-polyacrylamide gel electrophoresis. Cross-link formation required native Ung and was inhibited by increasing concentrations of NaCl in a manner characteristics of NaCl inhibition of Ung catalytic activity. The Ung x dT20 complex was purified to apparent homogeneity, and mass spectrometry revealed that Ung was cross-linked to dT20 in 1:1 stoichiometry as a 31,477 dalton complex. Purified Ung x dT20 lacked detectable uracil-DNA glycosylase activity and failed to bind single-stranded DNA. Recently, we demonstrated that the bacteriophage PBS2 uracil-DNA glycosylase inhibitor (Ugi) binds Ung and prevents further interaction with DNA (Bennett, S. E., Schimerlik, M. I., and Mosbaugh, D. W. (1993) J. Biol. Chem. 268, 26879-26885). Addition of the Ugi protein to the cross-linking reaction blocked formation of the Ung x dT20 cross-link. Conversely, the Ung x dT20 cross-link was refractory to Ugi binding. Upon trypsin digestion of Ung x dT20, four distinct products were identified as peptide x dT20 cross-links. A combination of amino acid sequence and mass spectrometric analysis revealed that four tryptic peptides (T6, T18, T19, and T18/19) were adducted to dT20. These observations suggest that dT20 is cross-linked to the Ung DNA-binding site. |
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| Keywords |
Amino Acid Sequence; Bacteriophages/enzymology; Binding Sites; Cross-Linking Reagents; DNA Glycosylases; Escherichia coli/enzymology; Mass Spectrometry; Molecular Sequence Data; N-Glycosyl Hydrolases/chemistry; N-Glycosyl Hydrolases/metabolism; N-Glycosyl Hydrolases/radiation effects; Oligodeoxyribonucleotides/chemistry; Oligodeoxyribonucleotides/metabolism; Oligodeoxyribonucleotides/radiation effects; Peptide Fragments/chemistry; Poly T/chemistry; Poly T/metabolism; Poly T/radiation effects; Sequence Analysis; Ultraviolet Rays; Uracil-DNA Glycosidase |
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