Visualising the kinetics of dissociation of actinomycin from individual sites in mixed sequence DNA by DNase I footprinting

Fletcher, Michael C.; Fox, Keith R.

doi:10.1093/nar/21.6.1339

Cited by 16 publications

(19 citation statements)

References 31 publications

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“…In contrast, dissociation from (AT),,CCGC(AT),, is much slower, consistent with SDS dissociation studies 124-271 which showed that CGCA is a better binding site than GGCA. The latter sequence is also present in tyrT DNA and shows a dissociation time of about 30 min [31]. Studies on DNA fragments containing GpC sites flanked by (GT),, .…”

Section: Discussionmentioning

confidence: 97%

“…We have recently developed a modification of the footprinting technique for examining the dissociation of ligands from individual binding sites on a mixed-sequence DNA [31]. In this technique, a complex between the ligand and a radiolabelled DNA fragment is dissociated by adding a vast excess of unlabelled calf thymus DNA.…”

Section: Introductionmentioning

confidence: 99%

“…Firstly, even if the detergent does not interact with the complex, it makes a significant contribution to the ionic strength (2% SDS, masshol., corresponds to I = 0.08). Secondly, it can only provide average dissociation parameters for natural DNAs which contain a large number of potential sites.We have recently developed a modification of the footprinting technique for examining the dissociation of ligands from individual binding sites on a mixed-sequence DNA [31]. In this technique, a complex between the ligand and a radiolabelled DNA fragment is dissociated by adding a vast excess of unlabelled calf thymus DNA.…”

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confidence: 99%

“…The dissociation is visualised by the rate of disappearance of the footprinting pattern. Using this technique, we have shown that actinomycin dissociates from each of its binding sites in tyrT DNA at different rates [31] and have examined the dissociation of a synthetic quinoxaline antibiotic from its TpA-binding sites [32]. In this paper, we have used the modified footprinting technique to examine the dissociation of actinomycin from GpC sites which are located in different sequence environments.…”

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confidence: 99%

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Dissociation Kinetics of Actinomycin D from Individual GpC Sites in DNA

Fletcher

Fox

1996

European Journal of Biochemistry

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We have examined the kinetics of dissociation of actinomycin from GpC sites in several DNA fragments containing synthetic DNA inserts, by a variation of the footprinting technique. Complexes of the ligand with radiolabelled DNA fragments were dissociated by adding a large excess of unlabelled calf thymus DNA. Samples were removed from this mixture at subsequent time intervals and subjected to DNase I footprinting. The rate of disappearance of the footprints varied considerably between the GpC sites located in different sequence environments. Actinomycin dissociates more slowly from GpC sites flanked by (AT),, than A,, . T!<. Within regions of alternating AT, TGCA represents a better binding site than AGCT, and CGCA is a better binding site than GGCA. GpC sites flanked by (AC),, . (GT), present good binding sites; in this context, dissociation from CGCG is faster than from TGCA. Several studies have shown that the dissociation of actinomycin from DNA is slow, and may be an important factor for determining its biological activity [2]. Although the dissociation from synthetic DNAs, such as poly(dG-dC), is completely described by a single exponential [IS, 161, the dissociation from mixed-sequence DNAs is a complex reaction which requires at least three exponentials for its complete description, with time constants ranging over 10-1000s [2,[15][16][17][18]. This has been explained by suggesting that the multiexponential decay represents parallel dissociation of the drug from different binding sites on the heterogeneous DNA lattice, each of which possesses its own characteristic dissociation rate constant. A similar effect has been observed for the quinoxaline group of antibiotics which bind around the sequence CpG by bifunctional intercalation [19, 201. In contrast, the derivative actinomine, which lacks the peptide portion, binds to DNA with nearly the same affinity as actinomycin, yet displays much faster dissociation kinetics and has little biological activity [2]. KeywordsAlthough the fastest dissociating components must represent the interaction of the ligand with weaker sites, it is not clear whether these correspond to non-GpC sites or to a subset of GpC sites with lower affinity. Indeed, the dissociation from poly(dG-dC) is faster than the slowest component from natural DNAs 1171, suggesting that blocks of alternating GC residues do not represent the best ligand-binding site. Various studies have shown that the binding of actinomycin to GpC is influenced by the nature of the surrounding base pairs. The strongest [23]. In a series of dissociation experiments with synthetic oligonucleotides, Chen [24-271 has shown that, in general, actinomycin dissociates faster from GC sites which are preceded by G or followed by C than from other sites such as AGCA, AGCG or CGCA. The dissociation from ATATGCATAT is unusually slow (t,,, = 3000 s), while GGCC represents one of the fastest dissociating GpC sites [25]. Other studies have shown that actinomycin binds well to several non-GpC sites [28]. In particular, the sequen...

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Section: Discussionmentioning

confidence: 97%

Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 99%

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confidence: 99%

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Dissociation Kinetics of Actinomycin D from Individual GpC Sites in DNA

Fletcher

Fox

1996

European Journal of Biochemistry

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“…We have recently developed a modification of the footprinting technique [20] for visualising the dissociation of ligands from individual binding sites in a mixed sequence DNA. In this technique a complex between the ligand and radiolabelled DNA is dissociated by addition of excess unlabelled DNA.…”

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confidence: 99%

Visualising the dissociation of sequence selective ligands from individual binding sites on DNA

Fletcher

Fox

1996

FEBS Letters

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We have used a modification of the footprinting technique to measure the dissociation of mithramycin, echinomycin and nogalamycin from their binding sites in a natural DNA fragment. Complexes with radiolabelled DNA were dissociated by addition of unlabelled DNA. Samples were removed at various times and subjected to DNase I digestion, and the rate of dissociation from each site was estimated from the time-dependent disappearance of the footprints. For echinomycin the slowest rate of dissociation is from ACGT, while the slowest site for mithramycin contains four contiguous guanines. The dissociation of nogalamycin is extremely slow, even from its weaker sites; the slowest rate was from ACGTA, which took longer than 4 h, even at 37 ° C.Key words." Echinomycin; Mithramycin; Nogalamycin; Dissociation that the complexity observed with natural DNA is due to the parallel dissociation from different binding sites, each of which has different microscopic kinetic constants.We have recently developed a modification of the footprinting technique [20] for visualising the dissociation of ligands from individual binding sites in a mixed sequence DNA. In this technique a complex between the ligand and radiolabelled DNA is dissociated by addition of excess unlabelled DNA. Samples are removed from the reaction mixture at various times and subjected to DNase I footprinting. Dissociation from each binding site is visualised as the time-dependent disappearance of the footprint. In this paper we use this technique to examine the dissociation of echinomycin, mithramycin and nogalamycin from tyrT DNA, and obtain the first estimates for the dissociation of these ligands from individual binding sites. Materials and methods

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Characterization of noncovalent complexes formed between minor groove binding molecules and duplex DNA by electrospray ionization-mass spectrometry

Galefn

Smith

1995

J. Am. Soc. Mass Spectrom.

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The noncovalent complex formed in solution between minor groove binding molecules and an oligonucleotide duplex was investigated by electrospray ionization-mass spectrometry (ESI-MS). The oligonucleotide duplex formed between two sequence-specific 14-base pair oligonucleotides was observed intact by ESI-MS and in relatively high abundance compared to the individual single-stranded components. Only sequence-specific A:B duplexes were observed, with no evidence of random nonspecific aggregation (i.e., A:A or B:B) occurring under the conditions utilized. Due to the different molecular weights of the two 14-base pair oligonucleotides, unambiguous determination of each oligonucleotide and the sequence-specific duplex was confirmed through their detection at unique mass-to-charge ratios. The noncovalent complexes formed between the self-complementary 5'-dCGCAAATTTGCG-3' oligonucleotide and three minor groove binding molecules (distamycin A, pentamidine, and Hoechst 33258) were also observed. Variation of several electrospray ionization interface parameters as well as collision-induced dissociation methods were utilized to characterize the nature and stability of the noncovalent complexes. The noncovalent complexes upon collisional activation dissociated into single-stranded oligonucleotides and single-stranded oligonucleotides associated with a minor groove binding molecule. ESI-MS shows potential for the study of small molecule-oligonucleotide duplex interactions and determination of small molecule binding stoichiometry.

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Visualising the kinetics of dissociation of actinomycin from individual sites in mixed sequence DNA by DNase I footprinting

Cited by 16 publications

References 31 publications

Dissociation Kinetics of Actinomycin D from Individual GpC Sites in DNA

Dissociation Kinetics of Actinomycin D from Individual GpC Sites in DNA

Visualising the dissociation of sequence selective ligands from individual binding sites on DNA

Characterization of noncovalent complexes formed between minor groove binding molecules and duplex DNA by electrospray ionization-mass spectrometry

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