Radiation-induced thymine base damage and its excision repair in active and inactive chromatin of HeLa cells.

The extent of production and excision repair of 5,6-dihydroxydihydrothymine type base (t') damage was determined in transcriptionally active and inactive chromatin of HeLa cells after exposure to 6.8 MeV electrons. It was observed that not only the yield but also rate of repair of t' products was greater in the active chromatin compared to the inactive chromatin of HeLa cells. The results strongly indicate that the conformation of chromatin is an important factor in determining the sensitivity to radiation damage and accessibility to enzymes required for repair of such damage.

Age associated alteration in DNA damage and repair capacity in Turbatrix aceti exposed to ionizing radiation.

Excision repair capacity was measured in young and old Turbatrix aceti (phylum Nematoda) following exposure to ionizing radiation. Both repair synthesis and removal of 5,6-dihydroxydihydrothymine type (glycol) base damage were quantitated. At least two-fold higher glycol levels were produced in the DNA of young than of old nematodes for the same radiation dose. Young worms also excised glycol damage more rapidly and completely than old worms. Both peak repair synthesis activity and completion of repair synthesis occurred at earlier times during post-irradiation incubation in young nematodes. The data indicate there is a significant age-associated difference in both the incidence and removal of ionizing radiation damage in T. aceti which is used as a model of the ageing process.

Age-related changes in the excision repair capacity of Turbatrix aceti.

Excision repair capacity was measured in the free-living nematode Turbatrix aceti, a model aging system. Excision repair was assessed by both repair synthesis activity and the actual removal of pyrimidine dimers from the genome. The young organisms removed lesions more rapidly and completely than the old for all fluences tested. Repair synthesis began and peaked earlier for young nematodes than old. The data consistently indicated a decline in DNA excision repair capacity with age in the nematode.

Production and excision repair of cyclobutane-type pyrimidine dimers in the DNA of Turbatrix aceti.

The production and removal of 254 nm ultraviolet-induced pyrimidine dimers was measured in the DNA of the free-living nematode Turbatrix aceti. Approximately 0.0035 per cent pyrimidine dimers are produced per J/m2. Following a fluence of 100 Jm2, approximately 50 per cent of the dimeric photoproducts were excised within 60 min. The number of pyrimidine dimers excised did not change with increasing U.V. fluence, indicating saturation of the U.V. repair system in T. aceti. The results indicate a highly efficient and selective repair system in Turbatrix aceti for dimeric photoproducts.

Effect of cordycepin(3'-deoxyadenosine) on excision repair of 5,6-dihydroxy-dihydrothymine-type products from the DNA of Micrococcus radiodurans.

Cordycepin(3'-deoxyadenosine), a nucleoside analog, has been shown to enhance radiation-induced cell killing. In an effort to elucidate the possible mechanism for enhancement of cell killing, the effect of cordycepin on the excision repair of radiation-induced 5,6-dihydroxy-dihydrothymine-type (t') products from the DNA of wild type Micrococcus radiodurans was investigated. The capacity of M. radiodurans to excise nondimeric (t') products from its DNA was significantly impaired after cordycepin treatment. The results suggest that the increased radiation sensitivity of cordycepin-treated cells could be due to alterations in cellular processes that repair DNA damage.

Excision of ultraviolet and gamma ray products of the 5,6-dihydroxy-dihydrothymine type by nuclear preperations of xeroderma pigmentosum cells.

Monomeric products of the 5,6-dihydroxy-dihydrothymine type are produced in the DNA by both ultraviolet and ionizing radiations. The capacity of nuclear preparations from normal and Xeroderma pigmentosum (XP) cells (Complementation groups A, B, C and D) to excise such products from ultraviolet or gamma-irradiated T7DNA was comparable and was independent of radiation induced strand breaks.

Selective excision of gamma ray damaged thymine from the DNA of cultured mammalian cells.

Three mammalian cell lines (WI-38, SV40-transformed WI-38 and Chinese hamster ovary) were exposed to high doses of 137-Cs gamma rays and their DNA analysed, following various periods of postirradiation incubation, for products of the 5,6-dihydroxy-dihydrothymine type. Within fifteen minutes of incubation at 37 degrees C 70 to 90 percent of these radiation products were removed from acid-precipitable material in all three cell lines. The amount of DNA degradation induced by radiation varied from approximately one percent in WI-38 cells to 15 percent in SV40-transformed WI-38 cells. Comparison of DNA degradation with the amount of thymine radiation product removed indicates that a selective gamma ray-induced excision repair capability exists in mammalian cells. Because of its more rapid kinetics, gamma ray excision repair is probably a distinct process as compared with ultraviolet-induced pyrimidine dimer excision.

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

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.

Excision of damaged thymine residues from gamma-irradiated poly(dA-dT) by crude extracts of Escherichia coli.

Crude extracts of E. coli endo I(-) and E. coli endo I(-)uvrA6(-) possess the ability to remove thymine products of the 5,6-dihydroxy-dihydrothymine type from gamma-irradiated or osmium tetroxide-oxidized poly(dA-dT). It is shown that the uvrA-gene product, which is responsible for incision close to photodimers in prereplication ultraviolet repair in E. coli, is not required for, but may aid in, the excision of gamma-ray products of the 5,6-dihydroxy-dihydrothymine type. Ring-damaged thymine products are also removed by E. coli extracts from osmium tetroxide-oxidized poly(dA-dT), which contains only 5,6-dihydroxy-dihydrothymine but no strand breakage, indicating that product excision occurs in the absence of radiation-induced breaks. On the average, 8 to 16 nucleotides are removed from the polymer per ring-damaged thymine residue excised by extracts from both strains and for gamma-irradiated and osmium tetroxide-oxidized polymer.