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Sequence Specific Cleavage of Double Helical DNA N-Bromoacetyldistamycin


Baker, Brenda Faye (1988) Sequence Specific Cleavage of Double Helical DNA N-Bromoacetyldistamycin. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/3c0t-vj26.


This thesis is concerned with the design, synthesis, and mechanism of interaction of sequence specific double helical DNA cleaving molecules. The design of sequence specific cleaving molecules for double helical DNA requires the attachment of DNA cleaving moieties to sequence specific DNA binding molecules. The degree of cleavage specificity is in part controlled by the cleavage moiety. With this in mind attention is focused on attachment of a nondiffusible cleavage moiety, specifically an electrophile, to a sequence specific binding unit. Electrophilic attack of DNA nucleophiles, such as the N-3 of adenine or the N-7 of guanine, results in cleavage of the backbone via depurination.

N-Bromoacetyldistamycin (BD) is designed to place an electrophile in the minor groove within adenine-thymine rich regions in close proximity to the N3 of adenine. The electrophile, bromoacetyl, is attached at the amino end of tris-N-methylpyrrolcarboxamide, a tripeptide binding unit from the natural product distamycin A.

A general synthetic scheme has been devised which allows for synthesis of the isotopically labeled compounds (carbon 13 and 14) and the other halide derivatives, iodo and chloro, for purposes of product characterization and kinetic analysis.

Affinity cleavage studies by high resolution gel electrophoresis show that BD has a high degree of cleavage specificity. Out of 334 bases in a 167 b.p. restriction fragment, from the pBR322 plasmid, cleavage occurs primarily (80%) at a single adenine after 10 hours at 37 degrees. In order to understand this high degree of specificity the mechanistic details of the reaction have been explored.

Under longer reaction times and higher temperatures cleavage occurs at other adenines of the 167 b.p. restriction fragment. The amount of cleavage at any given adenine is dependent upon the leaving group as shown with the hydroxy, chloro, iodo and mesyl derivatives, where Br > I ≃ Ms > Cl >>> OH.

Electrophilic attack of double helical DNA by BD occurs at the N3 of adenine. In this regard, carbon 14labeled BD was utilized to compare the products from the BD/free base adenine reaction and the BD/DNA reactions. 99% enriched [15N3]-adenine, 99% enriched N-Bromo-[2-13C]-acetyldistamycin, and subsequently the four isotopic combinations of the distamycin-adenine adduct were synthesized to determine the site of alkylation by proton, carbon 13, and nitrogen 15 NMR. The adenine adduct exists in the amino form (N6) as shown by NMR of the 15N6 labeled adduct.

3-(Acetyldistamycin)-adenine is the sole released product from the reaction of BD with a 15 base pair oligonucleotide, which contains the site of major cleavage from the 167 b.p. restriction fragment.

DNA products at each stage of the reaction and workup procedure were monitored by high resolution gel electrophoreses of radioactively labeled DNA. The alkylated DNA-distamycin intermediate has been detected. The DNA end products were determined enzymatically with phosphatases verifying that both the 3' and 5' DNA termini are phosphates as expected for an alkylation/depurination reaction followed by base workup.

The reaction of the N-haloacetyldistarnycins (Cl, Br, and I) with double helical DNA which results in strand cleavage has four experimentally distinguishable steps: 1) binding, 2) covalent attachment to adenine, 3) release of the adenine adduct, and 4) strand cleavage. Steps one through three were quantitatively analyzed using a synthetic oligonucleotide that contained the site of major cleavage (one binding site) of the 167 restriction fragment. In this system the binding constant for BD measures as K = 5.5 x 105 M-1. The rate of akylation is first order (k = 1.94 x 10-2 h-1, 37°C) and the rate of adenine adduct release is first order (7.76 x 10-2 h-1, 37°C).

The cleavage specificities of the synthetic molecule, N-Bromoacetyldistamycin, and the natural product, CC1065 were compared on the 167 b.p. restriction fragment. After a reaction time of one hour at 37°C, CC1065 cleaves at 21 adenines whereas BD shows only a minute amount of cleavage at one adenine.

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:
  • Roberts, John D. (chair)
  • Dervan, Peter B.
  • Richards, John H.
  • Dougherty, Dennis A.
Defense Date:23 May 1988
Additional Information:Thesis title listed in 1988 commencement program varies from actual thesis: Sequence-Specific Cleavage of Double-Helical DNA N-Bromoacetyldistamycin.
Record Number:CaltechTHESIS:01222013-095919666
Persistent URL:
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:7418
Deposited By: Dan Anguka
Deposited On:22 Jan 2013 18:39
Last Modified:16 Apr 2021 23:01

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