Sequence-Specific Inhibition of DNA Polymerase by Phenanthrene Quinone Diimine Complexes of Rhodium(III)

Citation

Johann, Timothy Wilmot (1997) Sequence-Specific Inhibition of DNA Polymerase by Phenanthrene Quinone Diimine Complexes of Rhodium(III). Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/fb9e-rw88. https://resolver.caltech.edu/CaltechTHESIS:08042025-205701232

Abstract

The DNA binding characteristics of several phenanthrenequinone diimine (phi) complexes of rhodium (III) as well as their ability to inhibit functionally DNA polymerase have been investigated. Affinity constants have been determined to be 5x10⁷ M^-1 and 1x10⁸ M^-1 for Δ and Λ 1Rh( MGP)₂phi⁵⁺ binding to the DNA sequences 5'-CATCTG-3' and 5'- CATATG-3' respectively. The exchange rate, at 21°C, has been determined to be 16 s^-1 for the binding of 1-Λ-Rh(MGP)₂phi⁵⁺ to 5'-CATATG-3' through the use of variable temperature ¹H-NMR. Similar ¹H-NMR experiments were carried out to determine the kinetics of the interaction of 1-Δ-Rh(MGP)₂phi⁵⁺ with a duplex DNA of the sequence 5'-CGCATCTGAC-3'. 1-Λ-Rh( MGP)₂phi⁵⁺, 1-Δ-Rh(MGP)₂phi⁵⁺, and Rh(MT)phi³⁺, which binds to 5'- TGCA-3', were found to be potent sequence-specific inhibitors of DNA polymerase. All of these complexes bind to DNA through intercalation. In experiments where two templates competed for extension by DNA polymerase, these complexes were shown to inhibit the extension of templates containing their binding sequences as compared to control templates. Furthermore, in direct competition experiments containing two templates, where each contained a binding sequence for a different metal complex, the relative activity of DNA polymerase on each template was "tuned" by the addition of metal complex specific for that template. Δ-Rh( DPB)2phi³⁺ was also found to be a potent inhibitor of DNA polymerase, but not in a template-specific manner. The relative potency of sequence- specific inhibition shown by 1-Λ-Rh(MGP)₂phi⁵⁺, 1-Λ-Rh(MGP)₂phi⁵⁺, and Rh(MT)₂phi³⁺ was compared to the binding kinetics, complex size, complex charge, binding affinity and binding induced DNA distortion for these complexes. Greater DNA distortion was found to correlate with greater inhibition. These studies have shown that these molecules not only bind to DNA in a sequence-specific manner, but can functionally inhibit enzymatic reactions in a sequence-specific manner as well.

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