Date of Award


Degree Type


Degree Name

Doctor of Philosophy


The buoyant density shift method was employed in the study of the enzymology and its regulation of DNA repair in cultured human cells. Measurements of the size of the DNA repair patch after treatments with UV-light or methylating agents revealed that about 30 nucleotides are replaced at each damage site, and that the extent of replacement is independent of the DNA-damaging agents employed.;In the examination of the role of poly(ADP-ribosyl)ation in DNA repair, the size of the repair patch was not increased by the presence of 3-aminobenzamide (3-AB), an inhibitor of poly(ADP-ribose) polymerase, as had been predicted by other investigators.;To elucidate the role of polymerase (alpha) in excision repair, aphidicolin was used to inhibit this enzyme specifically during the repair of DNA. Although there was no difference in the extent of repair-incorporation of the label between cells that performed DNA repair in the absence or in the presence of aphidicolin following UV-irradiation, the presence of the inhibitor produced repair patches that were at least twice that of normal. However, when DMS was used as the DNA-damaging agent, aphidicolin caused a decrease in the extent of repair replication and an accompanying increase in repair patch size.;On the basis of the study done with aphidicolin, several conclusions were drawn. Firstly, DNA polymerase (alpha) is involved in the repair of DNA. Secondly, the degree of incorporation of label into DNA following exposure to DNA-damaging agents may not always be a reliable way to quantitate repair events in the cell. Finally, it is proposed that the size of the repair patch is regulated by the relative rates of excision and polymerization in the cell.;It is further proposed, based on the known characteristics of (alpha) and (beta) polymerases, that the size of the single-stranded gap produced by the exonuclease determines the polymerase which fills the gap. A small gap of about 10 nucleotides in length, produced when the dose of UV-light is low, is filled in primarily by polymerase (beta). When the dose of UV-light is high, and the level of activity of the (beta) enzyme is insufficient to fill in the gaps, a larger gap of 30 nucleotides or more is produced. This is filled in partially by polymerase (alpha), and completed by the (beta) enzyme.



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