Different rate-limiting steps in excision repair of ultraviolet- and N-acetoxy-2-acetylaminofluorene-damaged DNA in normal human fibroblasts.

Abstract

In normal human cells the amount of excision of ultraviolet damage to DNA saturates at high doses. In these cells some chemicals mimic ultraviolet damage as far as their biological and repair characteristics are concerned. One of these chemicals is N-acetoxy-2-acetylaminofluorene. We determined whether the limited repair capacity for ultraviolet damage was affected by treatment with N-acetoxy-2-acetylaminofluorene. To Biological systems possess a number of enzymatic mechanisms to repair physical and chemical damage to their DNA (1-4). One of them-excision repair-involves four (sometimes five) general steps: (N-glycosidase action), incision, excision, polymerization, and ligation. This process acts upon a large number of chemically distinct damages induced by agents such as ultraviolet (UV), ionizing radiation, and a variety of chemical mutagens and carcinogens. Although it is reasonable that the versatility in substrate recognition for repair may be provided by a number of specific endonucleases each of which probably recognizes a class of distortion of the damaged site, the number of classes identified so far is small. A crude classification in terms of repair characteristics is into UV and ionizing radiation type (5).UV-induced pyrimidine dimers and N-acetoxy-2-acetylaminofluorene (AAAF) lesions in DNA are substrates for excision repair in human cells, and AAAF damage mimics UV damage in the following ways: (i) both are repaired by a long patch mechanism (about 100 nucleotides) (5); (ii) xeroderma pigmentosum (XP) cells deficient in repairing UV damage are also deficient in repairing AAAF damage (6, 7); (Mii) XP cells are more sensitive than normal cells to the cytotoxic and mutagenic activity of both UV and AAAF (8). However, studies based on DNA repair synthesis showed that removal of AAAF and UV lesions differed in the initial rate (9).The amount of excision repair of pyrimidine dimers saturates at high UV doses (>20 J-m-2 of 254 nm) (10-12). Hence, if AAAF truly mimicked UV damage one would expect that the amount of repair from a combined treatment using high doses would be less than the sum of the treatments separately. Therefore, we determined if the limited UV repair was affected by AAAF treatment. For this purpose we employed two techniques to monitor excision repair: unscheduled DNA synthesis (13) and the measurement of sites sensitive to UV-endonuclease (14). The first technique was chosen because it gives information of repair due to both agents, whereas the second, using Micrococcus luteus endonuclease, allows us to detect UVinduced pyrimidine dimers in the DNA of cells exposed to a combined treatment of UV and AAAF. We found, contrary to our expectation, that as a result of the combined treatment unscheduled DNA synthesis equaled the sum of those from separate treatments and that AAAF treatment did not inhibit the removal of sites sensitive to exogenous endonuclease. MATERIALS AND METHODSCell Line and Tissue Culture. The normal human fibroblasts Rid Mor CRL 1220 were obtained from the Am...

Quantitation of Pyrimidine Dimers by Immunoslot Blot Following Sublethal Uv‐irradiation of Human Cells

“…Human cells are highly proficient for the excision repair of pyrimidine dimers following U V irradiation. Most in vitro studies utilizing normal human skin fibroblasts in culture indicate 50% removal of pyrimidine dimers in 6 to 12 h (Gibson and D'Ambrosio, 1982;Gibson-D'Ambrosio et al, 1983;Konze-Thomas et al, 1979;Ahmed et al, 1977;Bohr et al, 1986) following exposures between 5 and 25 J m-2 U V radiation. Our studies using the ISB assay for dimer induction by 2 and 5 J m-2 U V radiation are consistent i.e.…”

“…Several methodologies have been used for the detection of pyrimidine dimers in irradiated cellular DNA (Freidberg andHanawalt, 1981, 1983). It has been established that normal human cells in vitro and in vivo exhibit a high level of excision repair of UV radiation induced pyrimidine dimers (Gibson and D'Ambrosio, 1982;Gibson-D'Ambrosio et al, 1983;Konze-Thomas et al, 1979;Ahmed et al, 1977;Bohr et al, 1986;Sutherland et al, 1980b;D'Amrosio et al, 1981a). Most of these studies require large number of cells, radiolabelled DNA and/or lethal doses of UV radiation.…”

Xeroderma pigmentosum exhibiting neurological disorders and systemic lupus erythematosus

“…The level of DNA repair was the same for both doses because this appears to be the range at which the rate of repair was saturated under these conditions, although others have found saturation to set in at doses slightly higher (Fig. 2) (Ahmed & Setlow 1977, Brown et al 1979. The XPlMI cells perform lower amounts of repair than normal cells at all doses and times.…”

Dna Excision Repair in the Very Uv‐sensitive Xeroderma Pigmentosum Complementation Group a Strain Xp4lo

“…Pyrimidine dimers were quantified by determining the number of sites sensitive to an endonuclease isolated from Micrococcus luteus (Carrier and Setlow, 1970;Paterson et a[., 1973). Our specific procedures and methods of calculations have been described (Ahmed and Setlow, 1977;Kantor and Setlow, 1981). Control cells labeled in DNA with I4C and UVirradiated cells labeled in DNA with 'H were mixed together at the appropriate post-UV incubation times and the DNAs coextracted immediately.…”