The interaction of mithramycin with DNA
The interaction of mithramycin with DNA
Mithramycin is an antitumour antibiotic which acts by binding to double stranded DNA. It is known to bind to GC rich DNA sequences requiring the presence of a divalent metal ion. The details of the interaction with DNA are uncertain, both major and minor grooves have been proposed for its binding site. In this thesis a variety of footprinting and spectral techniques have been used to elucidate its metal ion requirement, sequence specificity, mode of binding and effects on DNA structure. 1) Metal ion requirement. Spectrophotometric, fluorometric and footprinting studies have demonstrated that mithramycin requires divalent metal ions, particularly magnesium, to interact with DNA. The interaction of the drug with magnesium is weak (103M-1) and can not be substituted by calcium or barium. 2) Sequence selectivity. Footprinting studies on several DNA fragments using DNAaseI, hydroxyl radicals, and uranyl salts as probes have confirmed that the drug recognises GC rich regions of DNA. Most, though not all, of the binding sites contain the dinucleotide step GpG. Not all such sites are protected suggesting that binding is influenced by surrounding sequences. DNAaseI footprints also show regions of enhanced cleavage in the presence of the ligand, which are often located in adjacent runs of A and T. Footprinting with other enzymic probes have confirmed this selectivity and enhanced cleavage. 3) Structural changes. Chemical agents known to be sensitive to local DNA structure have been employed to investigate DNa structural changes induced by mithramycin. The lack of any changes with chemical probes acting via the major groove suggests the drug is located in the minor groove. Subtle changes in the reactivity of certain bases to diethylpyrocarbonate are consistent with the formation of an altered DNA structure around the drug binding site (more A-DNA like). The antibiotic does not affect the bending of kinetoplast DNA. The effect of mithramycin on local DNA structure was studied in more detail using several plasmids containing repetitive DNA sequences adjacent to good drug binding sites. Adjacent regions of (AT)n and (A)n revealed an altered nuclease cleavage pattern in the presence of drug. 4) Effects on nucleosome bound DNA. Footpringing studies, with reconstituted nucleosome cores have concluded that mithramycin binds to this DNA but does not alter its rotational positioning.
University of Southampton
Cons, Benjamin Major George
1990
Cons, Benjamin Major George
Cons, Benjamin Major George
(1990)
The interaction of mithramycin with DNA.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
Mithramycin is an antitumour antibiotic which acts by binding to double stranded DNA. It is known to bind to GC rich DNA sequences requiring the presence of a divalent metal ion. The details of the interaction with DNA are uncertain, both major and minor grooves have been proposed for its binding site. In this thesis a variety of footprinting and spectral techniques have been used to elucidate its metal ion requirement, sequence specificity, mode of binding and effects on DNA structure. 1) Metal ion requirement. Spectrophotometric, fluorometric and footprinting studies have demonstrated that mithramycin requires divalent metal ions, particularly magnesium, to interact with DNA. The interaction of the drug with magnesium is weak (103M-1) and can not be substituted by calcium or barium. 2) Sequence selectivity. Footprinting studies on several DNA fragments using DNAaseI, hydroxyl radicals, and uranyl salts as probes have confirmed that the drug recognises GC rich regions of DNA. Most, though not all, of the binding sites contain the dinucleotide step GpG. Not all such sites are protected suggesting that binding is influenced by surrounding sequences. DNAaseI footprints also show regions of enhanced cleavage in the presence of the ligand, which are often located in adjacent runs of A and T. Footprinting with other enzymic probes have confirmed this selectivity and enhanced cleavage. 3) Structural changes. Chemical agents known to be sensitive to local DNA structure have been employed to investigate DNa structural changes induced by mithramycin. The lack of any changes with chemical probes acting via the major groove suggests the drug is located in the minor groove. Subtle changes in the reactivity of certain bases to diethylpyrocarbonate are consistent with the formation of an altered DNA structure around the drug binding site (more A-DNA like). The antibiotic does not affect the bending of kinetoplast DNA. The effect of mithramycin on local DNA structure was studied in more detail using several plasmids containing repetitive DNA sequences adjacent to good drug binding sites. Adjacent regions of (AT)n and (A)n revealed an altered nuclease cleavage pattern in the presence of drug. 4) Effects on nucleosome bound DNA. Footpringing studies, with reconstituted nucleosome cores have concluded that mithramycin binds to this DNA but does not alter its rotational positioning.
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Published date: 1990
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Local EPrints ID: 460536
URI: http://eprints.soton.ac.uk/id/eprint/460536
PURE UUID: eb88fc6d-974f-4b79-a073-827c1b275250
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Date deposited: 04 Jul 2022 18:24
Last modified: 04 Jul 2022 18:24
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Author:
Benjamin Major George Cons
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