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Site specific mutagen reactions leading to the formation of adducts in DNA

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Item Summary

Title: Site specific mutagen reactions leading to the formation of adducts in DNA
Authors: Premaratne, Shyamal
Issue Date: 1992
Abstract: Several groups of chemicals are known to react with DNA. Some of these are known mutagens while others are potential carcinogens. In this work, an attempt was made to observe if such reactions were random or if indeed there are preferred bases / sequences where adduct formation occurs. A simple model was used for this work in which the DNA chain synthesis is terminated when the Sequenase™ enzyme reaches an adduct. The DNA used was M13mp18 (7.30 kilobases). This DNA is available from commercial sources in either the single or the double stranded circular form. The reactions were carried out with chloroacetaldehyde, or with the methyl, ethyl and propyl derivatives of N'-nitro-N-nitrosoguanidine, or with 2,4-dinitrophenylhydrazine, phenyl hydrazine, hydroxylamine hydrochloride, hydrazine hydrate or with methyl methanesulfonate. Position 81 (thymidine) was found to be a common fall off point .with all these chemicals. The other positions of fall off had some overlap but each chemical class had a unique adduct pattern. The methyl, ethyl and propyl derivatives of N'-nitro-N-nitrosoguanidine had the same positions of chain termination. Varying the salt concentration had a significant effect on the number of adducts formed when M13mp18 DNA was reacted with chloroacetaldehyde. A large number of termination points were observed in the presence of 100mM sodium acetate. Fluorescence spectroscopy was used to determine if in fact the chloroacetaldehyde was reacting with the DNA or if the mutagen merely prevented the enzyme from acting in the labeling step. It was found that the chloroacetaldehyde reacted with the DNA both in the single and the double stranded forms. Both the absorption and the fluorescence spectra indicated this and that nearly every base was adducted in most of the experiments reported here. Chloroacetaldehyde was also found to affect the Sequenase™ enzyme causing additional points of chain termination when it was added in the labeling step. Agarose gel electrophoresis showed that chloroacetaldehyde did not alter the migration pattern of either M13mp18 single or double stranded DNA or of the Hind III linear double stranded mixture. No evidence of hydrolysis was obtained. The ratio of supercoiled / relaxed forms of the double stranded M13mp18 DNA was not altered by the chloroacetaldehyde treatment. The adducted DNA sequenced in the same way as the non reacted but there were some 'blank' reactions in the sequencing lanes of the former.
Description: Thesis (Ph. D.)--University of Hawaii at Manoa, 1992.
Includes bibliographical references (leaves 134-147)
Microfiche.
xx, 147 leaves, bound ill. (some col.) 29 cm
URI/DOI: http://hdl.handle.net/10125/9376
Rights: All UHM dissertations and theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission from the copyright owner.
Appears in Collections:Ph.D. - Biomedical Sciences (Biochemistry)



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