Interactions between lac repressor protein and site-specific bromodeoxyuridine-substituted operator DNA. Ultraviolet footprinting and protein-DNA cross-link formation
- Rice Univ., Houston, TX (USA)
Specific contacts between the lac repressor and operator have been explored using 5-bromodeoxyuridine-substituted DNA. Substitution of BrdU for single thymidine positions in a synthetic 40-base pair operator provides substrate for ultraviolet irradiation; upon irradiation, strand scission occurs at the BrdU residues. When bound, lac repressor protein provides protection against UV-induced breakage depending on the nature of the sites and type of interaction. We have confirmed 13 unique sites of inducer-sensitive protection along the operator sequence using this method compared to complete substitution with BrdU; differences were observed at two positions for singly substituted versus completely substituted DNAs. The ability of these photosensitive DNAs to form short range cross-links to bound protein has been used to determine the efficiency with which cross-linked protein-DNA complexes are generated at each individual site of BrdU substitution. Five sites of high efficiency cross-linking to the repressor protein have been identified. At one site, cross-linking without protection from strand scission was observed; this result suggests an unusual mechanism of strand scission and/or cross-linking at this site. Comparison of the UV protection results and the cross-linking data show that these processes provide complementary tools for identifying and analyzing individual protein-DNA contacts.
- OSTI ID:
- 5574350
- Journal Information:
- Journal of Biological Chemistry; (USA), Vol. 266:10; ISSN 0021-9258
- Country of Publication:
- United States
- Language:
- English
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DNA
GENETIC RADIATION EFFECTS
STRAND BREAKS
RADIOINDUCTION
CROSS-LINKING
ESCHERICHIA COLI
GENE OPERONS
GENE REPRESSORS
REAGENTS
ULTRAVIOLET RADIATION
BACTERIA
BIOLOGICAL EFFECTS
BIOLOGICAL RADIATION EFFECTS
CHEMICAL REACTIONS
ELECTROMAGNETIC RADIATION
GENETIC EFFECTS
MICROORGANISMS
NUCLEIC ACIDS
NUCLEOPROTEINS
ORGANIC COMPOUNDS
POLYMERIZATION
PROTEINS
RADIATION EFFECTS
RADIATIONS
560120* - Radiation Effects on Biochemicals
Cells
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