I. Quantitation and analysis of furocoumarin:DNA adducts. II. Introduction and recovery of DNA in bacterial cells by electroporation

This thesis describes several new techniques for the quantitation and characterization of DNA adducts, and a high efficiency transfection system for analyzing the processing of damaged DNA in cells.;My method for measuring DNA interstrand crosslinks involves sedimenting the DNA through a sucrose gradient containing regions which sequentially denature the DNA with alkali, neutralize it, enzymatically digest single strand molecules that have not re-annealed, and separate double stranded molecules from digestion fragments. The number of interstrand crosslinks is related to the fraction of DNA that remains double stranded. I have assayed interstrand crosslinks produced in DNA by furocoumarins after exposure to long wavelength ultraviolet light in both bacterial and mammalian cells.;By analyzing the results of this method using a computer model I have shown that a single crosslink is insufficient to facilitate the rapid and complete renaturation of DNA molecules larger than about 20 million daltons. Evidence is presented that calcium dependent electrostatic interstrand crosslinks formed during the alkaline denaturation are responsible for this. The effect may be prevented by carrying out the denaturation in an alternating 50 gauss 60 Hz magnetic field.;I have developed a method for assaying bulky DNA adducts involving enzymatic hydrolysis of DNA to the mononucleotide level, followed by electrophoretic separation of nucleosides and adducted nucleosides and quantitation by absorption or radioactivity. This method was used to quantitate many kinds of adducts, including furocoumarin photoadducts, both in vivo and in vitro. I have also used this method, in addition to high pressure liquid chromatography, to investigate the photoadducts produced by 8-methoxypsoralen with cytosine in bacterial DNA and in oligonucleotides of defined sequence.;A new technique for introducing plasmid DNA into bacterial and other cells based on electroporation is described. It has been used to transform bacteria with high efficiency, and to insert up to several percent of the plasmid DNA in a solution into bacterial cells so that the processing of the population of molecules may be studied as a whole.