Salt dependence of calicheamicin-DNA site-specific interactions.
Calicheamicin gamma 1I site-specifically binds and cleaves three closely spaced tetranucleotide sequences embedded in an AT-rich region of a 142 base pair DNA restriction fragment. Cleavage is observed predominantly at the TCCT, TTGT, and ATCT sequences, of which TCCT is the primary cleavage site. The Gibbs free energies required to bind calicheamicin to these sequences within the DNA restriction fragment have been determined as a function of NaCl concentration at pH 8.1 and 0 degrees C and at pH 7.5 and 23 and 0 degrees C. Between 150 mM and 1 M NaCl, calicheamicin binding to all three sequences is insensitive to salt. The insensitivity of calicheamicin binding to salt continues to 50 mM NaCl for the TTGT and ATCT sequences; the delta G values for calicheamicin binding to these sequences are on the order of -7.8 to -7.9 kcal mol-1 over the entire range of NaCl concentrations studied. However, between 150 and 125 mM NaCl, the TCCT sequence displays a sharp transition in the delta G of calicheamicin binding from -7.6 to -8.9 kcal mol-1. Below 125 mM NaCl, the delta G values for calicheamicin binding to the TCCT sequence again are invariant. An analysis of the data in terms of polyelectrolyte theory suggests that counterion release from DNA does not contribute significantly to the energetics of the association and that the association of calicheamicin with specific DNA sequences is dominated by nonionic rather than electrostatic forces. Our results further suggest that some calicheamicin binding/cleavage sites are dependent on flanking sequences.
Krishnamurthy
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Natural Products Research Section, American Cyanamid Co., Lederle Laboratories, Pearl River, New York 10965.
Brenowitz
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Ellestad
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GA
D000617:Aminoglycosides; D000900:Anti-Bacterial Agents / Q000737:chemistry*; D000970:Antineoplastic Agents / Q000737:chemistry*; D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D004249:DNA Damage; D053281:Enediynes; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D010957:Plasmids; D012965:Sodium Chloride / Q000737:chemistry; D013816:Thermodynamics
D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D000970:Antineoplastic Agents; D053281:Enediynes; C055955:calicheamicin gamma(1)I; D012965:Sodium Chloride; D004247:DNA