The enzymic initiation of repair of photoalkylated bases in DNA will be investigated. An endonuclease activity that breaks such DNA will be purified from human placenta, cultured human cells and from Micrococcus luteus. These enzymes will be characterized. Detailed physical and chemical analysis of the substrate will be done to determine the precise type of DNA deformity recognized by the enzyme. The possible accumulation of damages to DNA in aging human tissues and cells will be investigated. DNA will be isolated from human organs obtained at autopsy and probed for sites using DNA repair enzymes. Senescent cells in culture will also be investigated in the same manner. The number of reiterated sequences in human DNA generated by reaction with Eco R1 endonuclease will be quantitatively analysed. Examination of brain, liver, muscle, spleen and kidneys for loss of the sequences and other types of DNA alterations will aid in the resolution of the problem of the role of cumulative DNA damage in cellular senescence. The activities of purified DNA repair enzymes acting on substrates with more than one form of DNA damage will be investigated. PBS-2 DNA, which contains uracil, will be used as substrate for uracil-DNA glycosylase. The effects of the presence of purine adducts of the carcinogens N-acetoxy-2-acetylaminofluorene and 4-nitroquinoline-1-oxide, 7-methylguanine and apurinic sites on the enzymatic excision of uracil from such DNA will be measured. Enzymological kinetic studies will be performed to elucidate the type and molecular basis of any inhibition of uracil excision caused by damaged DNA purines. The effects of the same damaged purines on the activity of the pyrimidine dimer-DNA glycosylase will be investigated. This enzyme initiates repair of ultraviolet-induced pyrimidne dimers. Left unrepaired, uracil in DNA is mutagenic and pyrimidine dimers are both mutagenic and carcinogenic. The DNA glycosylased to be investigated here initiate repair of both these important forms of DNA damage. In addition to elucidation of the mechanisms of activity of these important enzymes, this work will show how one form of chemical damge to DNA may exert its mutagenic or carcinogenic effect by interference with initiation of repair of a totally different type of DNA damage.
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