Date of Award


Degree Type


Degree Name

Doctor of Philosophy


Base excision repair of DNA and factors that affect DNA incision in human cells were considered in this study.;Treatment of alkylated HeLa cells with 3-aminobenzamide, and inhibitor of poly(ADP-ribose) polymerase, increased the number of DNA strand breaks but did not slow down their rejoining by DNA repair. Therefore, an increase in DNA incision, not a decrease in ligation, results from the inhibition of poly(ADP-ribose) polymerase in alkylated cells. This is consistent with recent findings by others that suggest that repair-patch frequency is increased in alkylated cells when polyADP-ribosylation is inhibited.;Apurinic/apyrimidinic (AP) sites were measured in HeLa cells by digestion of cellular DNA with Escherichia coli endonuclease IV, an AP-specific endonuclease, prior to alkaline elution. The absence of non-specific endonuclease activity allowed endonuclease IV-sensitive AP sites to be detected with the sensitivity of conventional alkaline elution. Cells that were alkylated with dimethylsulfate, but not benzo(a)pyrene diol epoxide, contained AP sites that were repaired along with DNA single-strand breaks during a post-alkylation recovery period. In addition, alkali-labile sites (that become single-strand breaks in the presence of alkali) other than AP sites were resolved in dimethylsulfate-treated cells by comparing the rate of elution of endonuclease IV-digested DNA at pH 12.1 and pH 12.6. Alkali-labile sites were also observed in benzo(a)pyrene diol epoxide-treated cells. However, these alkali-labile sites were not sensitive to endonuclease IV, and hence are not AP sites.;According to the base excision repair model, the sequential action of DNA glycosylases and AP endonucleases is thought to initiate the repair of DNA base damage. This model was tested in {dollar}\gamma{dollar}-irradiated HeLa cells by endonuclease IV-coupled alkaline elution. AP sites were detected as a transient DNA repair intermediate in {dollar}\gamma{dollar}-irradiated cells. This approach illuminated the operation of base excision repair in human cells by demonstrating the transit of ionizing radiation-induced base lesions, most of which are unknown, through this pathway.



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