Excision-repair of gamma-ray-damaged thymine in bacterial and and mammalian systems.

Abstract

The selective excision of products of the 5,6-dihydroxy-dihydrothymine type (t') from gamma-irradiated or OSO4-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 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' in equilibrium 3' exonuclease associated with polymerase I is responsible for the removal of t'. (4) Experiments with extracts of E. coli endo I lig 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 gamma-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.