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Chemical and Enzymatic Footprinting of Quinoxaline Antibiotics on DNA


Mendel, David (1990) Chemical and Enzymatic Footprinting of Quinoxaline Antibiotics on DNA. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/1kmd-qq48.


A key element of antitumor drug design is an understanding of how naturally occurring antitumor antibiotics recognize and bind to DNA, the major means by which many of these agents act. Once such an understanding is attained, the salient elements of recognition may be extracted and then extended to create more powerful and more specific antitumor (chemotherapeutic) agents. In an effort to understand G•C recognition, we studied the binding interactions between DNA and the antitumor antibiotics triostin A and echinomycin.

Recent studies by the Rich group at MIT and by the Patel group at Columbia have shown that the naturally occurring antitumor antibiotics triostin A and echinomycin bind four base pairs of DNA and can induce the formation of Hoogsteen base pairs at the first and fourth base pair positions of their binding sites on small oligonucleotides. The central aim of the thesis work described below was to establish whether this novel base-pairing occurs at echinomycin and triostin A binding sites on native DNA and if so, whether this represents a new recognition motif on which to base anticancer drug design.

We find that purines occupying the first and/or fourth base pair positions of echinomycin and triostin A binding sites become hyperreactive to diethyl pyrocarbonate (DEP) in the presence of drug. This finding raised the issue as to whether DEP detects Hoogsteen base-pairing at echinomycin binding sites in solution. We analyzed the products of DEP-purine reaction formed in the presence of echinomycin and ethidium bromide at six different echinomycin binding sites. Two different DEP-purine adducts were identified: 5-carbethoxyamino-4,6-diaminopyrimidine and 5-carbethoxyamino-2,4-diamino-6-hydroxypyrimidine. These products correspond to reaction of DEP at the N7 positions of adenosine and guanosine residues, respectively. The identity of these compounds strongly suggests that DEP responds to local helix unwinding caused by antibiotic intercalation into DNA and not to a Watson-Crick to Hoogsteen base-pairing transition caused by echinomycin binding to DNA. Nonetheless, DEP is a sensitive and precise probe of echinomycin and triostin A binding to DNA, and we describe this chemical footprinting reagent as the preferred means by which to identify echinomycin and triostin A binding sites on DNA. We also note three new types of echinomycin binding sites (5'-3') GGGG, TCAT, and TCAC not previously identified.

We find that another chemical footprinting reagent, dimethyl sulfate (DMS), demonstrated that isolated C.G Hoogsteen base pairs may exist at echinomycin binding sites within a DNA duplex under acidic conditions. If this is indeed the case, it would be the first example of isolated Hoogsteen base pairs to exist within a DNA fragment in solution. However, at neutral pH, echinomycin and triostin A appear to bind DNA via simple bisintercalation and probably do not form stable Hoogsteen base pairs at their binding sites. Therefore, we conclude that the induction or recognition of Hoogsteen base pairs at drug binding sites does not yet constitute a practical approach to the design of sequence specific DNA binding agents.

In addition, using a series of plasmid DNAs and a battery of enzymatic and chemical probes of DNA structure, we find that echinomycin binding to DNA apparently alters the helix structure of sequences adjacent and distal to its binding sites. Echinomycin can thus be considered an allosteric effector of DNA structure.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:Chemistry
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemistry
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Dervan, Peter B.
Thesis Committee:
  • Campbell, Judith L. (chair)
  • Dervan, Peter B.
  • Grubbs, Robert H.
  • Myers, Andrew G.
Defense Date:1 June 1989
Record Number:CaltechETD:etd-06072007-085143
Persistent URL:
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:2502
Deposited By: Imported from ETD-db
Deposited On:15 Jun 2007
Last Modified:23 Aug 2021 21:12

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