Identification of preferred actinomycin-DNA binding sites by the combinatorial method REPSA

Citation
J. Shen et al., Identification of preferred actinomycin-DNA binding sites by the combinatorial method REPSA, BIO MED CH, 9(9), 2001, pp. 2285-2293
Citations number
29
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Chemistry & Analysis
Journal title
BIOORGANIC & MEDICINAL CHEMISTRY
ISSN journal
0968-0896 → ACNP
Volume
9
Issue
9
Year of publication
2001
Pages
2285 - 2293
Database
ISI
SICI code
0968-0896(200109)9:9<2285:IOPABS>2.0.ZU;2-7
Abstract
An important question in the study of ligand-DNA interactions is the determ ination of binding specificity. Here, we used the combinatorial method rest riction endonuclease protection, selection, and amplification (REPSA) to id entify the preferred duplex DNA-binding sites of the antineoplastic agent a ctinomycin D. After 10 rounds of REPSA, over 95% of the cloned DNAs exhibit ed significantly reduced FokI restriction endonuclease cleavage in the pres ence of 1 muM actinomycin. A chi (2) statistical analysis of their sequence s found that 39 of the 45 clones contained one or more copies of the sequen ce 5 '-(T/A)GC(A/T)-3 ', giving a p <0.001 for this consensus. A DNase I fo otprinting analysis of the cloned DNAs found that all possessed relatively high affinity actinomycin-binding sites with apparent dissociation constant s 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 consen sus sequences (e.g., 5 ' -TGCTGCT-3 '), suggesting that such close proximit y DNA-binding sites may actually be preferred by actinomycin under physiolo gical conditions. (C) 2001 Elsevier Science Ltd. All rights reserved.