Hydroxyl radical footprinting of calicheamicin. Relationship of DNA binding to cleavage.
The binding to DNA by calicheamicin epsilon (CLM epsilon), the rearranged and reduced product of the diynene antitumor antibiotic calicheamicin gamma 1I (CLM gamma 1I), was studied using the method of hydroxyl radical footprinting. The drug binding sites determined in this way were compared to locations of double-stranded DNA cleavage by thiol-activated CLM gamma 1I. The results of these experiments show that CLM epsilon lies in the minor groove in an extended conformation protecting approximately four nucleotides on each strand of DNA. Sites of CLM epsilon binding correlate to sites of CLM gamma 1I cleavage with protection by CLM epsilon occurring mainly to the 3' side of the site of C5' hydrogen abstraction. From these results, it is possible to propose global structures of the drug/DNA complexes such that the oligosaccharide side chain is arrayed to the 3' side of the site of C5' hydrogen abstraction. This conclusion is entirely consistent with the results of recent atom-transfer experiments [Hangeland, J.J., De Voss, J.J., Heath, J.A., Townsend, C.A., Ding, W.-D., Ashcroft, J., & Ellestad, G.A. (1992) J. Am. Chem. Soc. 114, 9200-9202]. Somewhat greater protection on the strand undergoing C5' hydrogen abstraction was observed to the 5' side of the site of attack owing presumably to proximity of the methyl carbamate portion of the drug with DNA. Overall, binding is seen where cleavage is seen in accord with thermodynamics of drug association to DNA being important in determining the sites of cleavage.
Mah
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Department of Chemistry, Johns Hopkins University, Baltimore, Maryland 21218.
Townsend
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Tullius
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D000617:Aminoglycosides; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism*; D000903:Antibiotics, Antineoplastic / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D015246:Deoxyribonuclease EcoRI / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D053281:Enediynes; D017665:Hydroxyl Radical; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D015394:Molecular Structure; D013816:Thermodynamics
D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D000903:Antibiotics, Antineoplastic; D053281:Enediynes; C055955:calicheamicin gamma(1)I; C085353:calicheamicin epsilon; D017665:Hydroxyl Radical; D004247:DNA; D015246:Deoxyribonuclease EcoRI; D015252:Deoxyribonucleases, Type II Site-Specific; C046719:GGCGCC-specific type II deoxyribonucleases