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Identification of preferred actinomycin–DNA binding sites by the combinatorial method REPSA by Jing Shen; Jo C. Wang; Michael W. Van Dyke is a Biochemistry, Genetics and Molecular Biology article available to read on EtoBox.

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An important question in the study of ligand-DNA interactions is the determination of binding specificity. Here, we used the combinatorial method restriction endonuclease protection, selection, and amplification (REPSA) to identify the preferred duplex DNA-binding sites of the antineoplastic agent actinomycin D. After 10 rounds of REPSA, over 95% of the cloned DNAs exhibited significantly reduced FokI restriction endonuclease cleavage in the presence of 1 mM actinomycin. A w 2 statistical analysis of their sequences found that 39 of the 45 clones contained one or more copies of the sequence 5 0 -(T/A)GC(A/T)-3 0 , giving a p <0.001 for this consensus. A DNase I footprinting analysis of the cloned DNAs found that all possessed relatively high affinity actinomycin-binding sites with apparent dissociation constants between 12 and 258 nM (average 98 nM). The average footprint encompassed 7.6 bases and in most cases (90%) included one or more consensus sequences. Interestingly, several of the selected clones contained overlapping consensus sequences (e.g., 5 0 -TGCTGCT-3 0 ), suggesting that such close proximity DNA-binding sites may actually be preferred by actinomycin under physiologi

Who reads Identification of preferred actinomycin–DNA binding sites by the combinatorial method REPSA?

Author

Jing Shen; Jo C. Wang; Michael W. Van Dyke

Publisher

Elsevier Science; Elsevier ; Elsevier Ltd.; Elsevier BV (ISSN 0968-0896)

Published

2001

Language

EN

Field

Biochemistry, Genetics and Molecular Biology (Life Sciences)