Student Theses and Dissertations

Date of Award

1967

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Thesis Advisor

Edward Reich

Keywords

copolymer, thymine, actinomycin, RNA polymerase, 2-aminopurine

Abstract

An alternating copolymer of deoxy 2,6 diamino purine and thymine (dDAP-T) has been synthesized by substituting 2,6 diamino purine deoxyriboside triphosphate for dATP in a dAT primed synthesis with DNA polymerase. This polymer differs from dAT in having an amino group in the minor groove, and in several physical properties. For example, the additional amino group allows a third hydrogen bond of the Watson-Crick type to be formed with thymine, and the expected increase in the stability of the helix is reflected in a transition temperature (Tm) which is higher by 25°C. than that of dAT. Also, the buoyant density of dDAP-T in CsCl differs appreciably (ρ = 1.718) from that of dAT (ρ = 1.679). The antibiotic actinomycin has been shown to inhibit RNA polymerase by binding to helical DNA. The specificity of this interaction is thought to be determined by the 2-amino group of guanine since neither dAT nor dI:dC interact with the antibiotic. dAT-like polymers containing 2,6 diaminopurine can be shown to interact with actinomycin by the following criteria: 1) The buoyant density of the polymer in CsCl gradients is altered in the presence of the antibiotic. 2) The Tm of the dAT-like polymers containing DAP is increased in the presence of actinomycin. 3) The visible spectrum of actinomycin is changed in the presence of dDAP-T. 4) The template function of dAT-like polymers containing DAP for RNA polymerase is strongly inhibited by actinomycin. Thus the introduction of an amino group at the 2 position of the purine in the minor groove of helical DNA suffices to convert the A-T base pair from antibiotic indifference to antibiotic sensitivity. The second purine analogue studied in this thesis is 2-aminopurine. Like 2,6 diaminopurine, 2-aminopurine is a substrate analogue of only dATP with DNA polymerase. Also dAT polymers containing 2AP are found to interact with actinomycin. However, the substrate properties and the physical characteristics of polynucleotides containing 2-aminopurine are unusual. 1) Significant utilization of d2APTP by DNA polymerase with all templates (exonuclease III-treated DNA, dAT, dA:dT, dAC:dTG) tested requires the simultaneous presence of dATP. 2) The thermal denaturation profile of dAT (12.5% 2AP) is non-cooperative, and occurs at a lower temperature than dAT. 3) The degree of hyperchromicity of polymers containing 2AP is appreciably lower than that of dAT. To explain these unusual properties, it is proposed that the 2AP-T base pair is fully denatured under the conditions used for enzymatic synthesis and for the study of polymers containing 2AP. A twofold theory to explain the mutagenic action of 2-aminopurine is as follows: 1) Since the 2AP-T base pair is believed to be denatured, the presence of this unwound segment of DNA corresponds to the events which follow U.V. irradiation of DNA. The mutation occurs as a result of a mistake during the course of repair. 2) At an acid pH the N-1 of dCTP is protonated. Therefore, a 2AP-C base pair containing two hydrogen bonds can be formed at an acid pH. Since the pH of C in polynucleotides is displaced to lower pH values than that of the monomer, 2AP would be expected to pair with protonated dCTP more frequently than d2APTP pairing with a protonated C residue in the template DNA. These hypotheses are currently under investigation.

Comments

A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy

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