Excision-Repair of γ-Ray-Damaged Thymine in Bacterial and Mammalian Systems

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Abstract
The selective excision of products of the 5,6-dihydroxy-dihydrothymine type (t′) from γ-irradiated or 0s04-oxidized DNA or synthetic poly[d(A-T)] was observed with crude extracts of Escherichia coli and isolated nuclei from human carcinoma HeLa S-3 cells and Chinese hamster ovary cells. The results with E. coli extracts allow the following conclusion: (1) The uvrA-gene product is not required for t′ excision. (2) Radiation-induced strand breakage is not required for product excision. (3) Experiments with extracts of E. coli polAexl showed that the 5′→3′ exonuclease associated with polymerase I is responsible for the removal of t′. (4) Experiments with extracts of E. coli endo I rig 4 and the ligase inhibitor nicotinamide mononucleotide showed that polynucleotide ligase accomplishes the last strand resealing step in the excision-repair of t′. Isolated nuclei from HeLa and Chinese hamster ovary cells possess the necessary enzymes for the selective excision of t′ from γ-irradiated or osmium tetroxide oxidized DNA. Approximately 25 to 35% of the products were removed from DNA within 60 min. Unspecific DNA degradation was very low. Radiation-induced strand breakage is not required for product removal.

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