Interactions of photoactive DNAs with terminal deoxynucleotidyl transferase: identification of peptides in the DNA binding domain.
Terminal deoxynucleotidyl transferase (terminal transferase) was specifically modified in the DNA binding site by a photoactive DNA substrate (hetero-40-mer duplex containing eight 5-azido-dUMP residues at one 3' end). Under optimal photolabeling conditions, 27-40% of the DNA was covalently cross-linked to terminal transferase. The specificity of the DNA and protein interaction was demonstrated by protection of photolabeling at the DNA binding domain with natural DNA substrates. In order to recover high yields of modified peptides from limited amounts of starting material, protein modified with 32P-labeled photoactive DNA and digested with trypsin was extracted 4 times with phenol followed by gel filtration chromatography. All peptides not cross-linked to DNA were extracted into the phenol phase while the photolyzed DNA and the covalently cross-linked peptides remained in the aqueous phase. The 32P-containing peptide-DNA fraction was subjected to amino acid sequence analysis. Two sequences, Asp221-Lys231 (peptide B8) and Cys234-Lys249 (peptide B10), present in similar yield, were identified. Structure predictions placed the two peptides in an alpha-helical array of 39 A which would accommodate a DNA helix span of 11 nucleotides. These peptides share sequence similarity with a region in DNA polymerase beta that has been implicated in the binding of DNA template.
Farrar
Y J
YJ
Department of Biochemistry, University of Kentucky, Lexington 40536-0084.
Evans
R K
RK
Beach
C M
CM
Coleman
M S
MS
D000345:Affinity Labels; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents; D004247:DNA / Q000737:chemistry*; D004253:DNA Nucleotidylexotransferase / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D010777:Photochemistry; D011108:Polymers; D013698:Templates, Genetic
D000345:Affinity Labels; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D010455:Peptides; D011108:Polymers; D004247:DNA; D004253:DNA Nucleotidylexotransferase