UV-catalyzed cross-linking of Escherichia coli uracil-DNA glycosylase to DNA. Identification of amino acid residues in the single-stranded DNA binding site.
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.
Bennett
S E
SE
Department of Agricultural Chemistry, Oregon State University, Corvallis 97331.
Jensen
O N
ON
Barofsky
D F
DF
Mosbaugh
D W
DW
D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000201:enzymology; D001665:Binding Sites; D003432:Cross-Linking Reagents; D045647:DNA Glycosylases; D004926:Escherichia coli / Q000201:enzymology; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000737:chemistry / Q000378:metabolism / Q000528:radiation effects*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism / Q000528:radiation effects*; D010446:Peptide Fragments / Q000737:chemistry; D011071:Poly T / Q000737:chemistry / Q000378:metabolism / Q000528:radiation effects*; D017421:Sequence Analysis; D014466:Ultraviolet Rays; D051981:Uracil-DNA Glycosidase
D003432:Cross-Linking Reagents; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D011071:Poly T; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase