Interactions of photoactive DNAs with terminal deoxynucleotidyl transferase: Identification of peptides in the DNA binding domain

Journal Article · Tue Mar 26 04:00:00 EST 1991 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00226a014· OSTI ID:5596942

Farrar, Y J.K.; Evans, R K; Beach, C M; Coleman, M S ^[1]

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1. Univ. of Kentucky, Lexington (USA)

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{prime} 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 {sup 32}P-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 {sup 32}P-containing peptide-DNA fraction was subjected to amino acid sequence analysis. Two sequences, Asp{sup 221}-Lys{sup 231} (peptide B8) and Cys{sup 234}-Lys{sup 249} (peptide B10), present in similar yield, were identified. Structure predictions placed the two peptides in an {alpha}-helical array of 39 {angstrom} 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.

• OSTI ID: 5596942
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 30:12; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMINO ACID SEQUENCE
AMINO ACIDS
ASPARTIC ACID
BACTERIA
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CARBOXYLIC ACIDS
CHEMICAL REACTIONS
CROSS-LINKING
DAYS LIVING RADIOISOTOPES
DNA
ELECTROPHORESIS
ENZYMES
ESCHERICHIA COLI
HYDROGEN COMPOUNDS
ISOTOPES
LIGHT NUCLEI
LYSINE
MICROORGANISMS
MOLECULAR STRUCTURE
NUCLEI
NUCLEIC ACIDS
ODD-ODD NUCLEI
OLIGONUCLEOTIDES
ORGANIC ACIDS
ORGANIC COMPOUNDS
PEPTIDES
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
POLYMERIZATION
PROTEINS
RADIOISOTOPES
TRANSFERASES
TRITIUM COMPOUNDS