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

Author: Johann, Timothy Wilmot

Year: 1997

Degree: Dissertation (Ph.D.)

Advisor: Barton, Jacqueline K.

Committee Members: Baldeschwieler, John D.; Barton, Jacqueline K.; Okumura, Mitchio; Parker, Carl Stevens

Option: Chemistry

DOI: 10.7907/fb9e-rw88

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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