James D. Regan

Inhibition of DNA repair in ultraviolet-irradiated human cells by hydroxyurea

The effect on DNA repair in ultraviolet-irradiated human skin fibroblasts by hydroxyurea has been examined in this study using three independent methods for measuring DNA repair:the 5-bromodeoxyuridine photolysis assay which measures DNA repair replication, chromatographic measurement of thymine-containing dimers, and measurement of specific ultraviolet-endonuclease-sensitive sites in irradiated DNA. Little effect of hydroxyurea was observed at the concentration of 2 mM, which is often used to inhibit semiconservative DNA synthesis; however, 10 mM hydroxyurea resulted in marked inhibition (65--70%) of excision repair. This inhibition was accompanied by a possible doubling in the size of the repaired region. The accumulation of large numbers of single-strand breaks following ultraviolet irradiation and hydroxyurea incubation seen by other investigators was not observed with the normal skin fibroblasts used in this study. A comparison of hydroxyurea effects on the different DNA repair assays indicates inhibition of one step in DNA repair also results in varying degrees of inhibition of other steps as well.

The relationship of DNA excision repair of ultraviolet-induced lesions to the maximum life span of mammals

Physical and chemical agents present in the environment can potentially damage mammalian DNA. Such damage is known in some cases to be repaired by the process of DNA excision repair. This process has been extensively studied utilizing the repair of ultraviolet irradiation damage as a model system. In this study we have used this system and the 5-bromodeoxyuridine photolysis assay to measure DNA excision repair in cells derived from 21 mammalian species. We have attempted to relate the DNA repair proficiencies and the average size of the repaired regions seen in the cell cultures with the various maximum life spans of the mammals studied. There was an approximate linear correlation between life span of the mammals and the number of DNA excision repair sites measured 20-22 hours following ultraviolet irradiation of the cell cultures. Several deviations from the linear relationships were observed which remain largely unexplained. The size of the repaired regions was shown not to be related to the maximum life spans of the mammals tested.

Steps in DNA Chain Elongation and Joining after Ultra-violet Irradiation of Human Cells

SummaryThe DNA synthesized in human cells shortly after u.v.-irradiation is of lower molecular weight than that in unirradiated cells. Within several hours after irradiation, these smaller DNA units are both elongated and joined together. To determine if this process involves incorporation of exogenous precursors, cells were irradiated, pulse-labelled, and incubated in medium containing bromodeoxyuridine. They were then exposed to different fluxes of 313-nm radiation, and the number of breaks in the DNA was determined by sedimentation in alkaline sucrose. If bromodeoxyuridine, rather than exclusively premade DNA, is used to elongate and join the small DNA segments synthesized after u.v.-irradiation, photolysis by 313-nm radiation would yield the lower molecular weight segments that are present immediately after u.v.-irradiation. This expectation was fulfilled, and our results indicate that the mechanism of filling the gaps in DNA synthesized after irradiation involves the insertion of ∼ 103 nucleotides pe...

DNA Chain Elongation and Joining in Normal Human and Xeroderma Pigmentosum Cells after Ultraviolet Irradiation

DNA synthesized in human cells after ultraviolet (UV) irradiation is made in segments of lower molecular weight than in unirradiated cells. Within several hours after irradiation these smaller units are both elongated and joined together. This repair process has been observed in normal human fibroblasts, HeLa cells, and fibroblasts derived from three types of xeroderma pigmentosum patients-uncomplicated with respect to neurological problems, complicated (de Sanctis-Cacchione syndrome), and one with the clinical symptoms of xeroderma pigmentosum but with normal repair replication. The ability of human cells to elongate and to join DNA strands despite the presence of pyrimidine dimers enables them to divide without excising the dimers present in their DNA. It may be this mechanism which enables xeroderma pigmentosum cells to tolerate small doses of UV radiation.

Defective repair of N-acetoxy-2-acetylaminofluorene-induced lesions in the DNA of xeroderma pigmentosum cells*

Summary Cells from normal individuals and from individuals with xeroderma pigmentosum were treated with the carcinogen N-acetoxy-2-acetylaminofluorene. Repair of the damage to DNA was estimated by incubating the cells in BrdUrd and then photolyzing the BrUra-containing repaired regions with 313-nm radiation. By this criterion normal cells repair the damage in a fashion similar to ultraviolet damage, but xeroderma pigmentosum cells (defective in ultraviolet repair) are defective in repair of the carcinogen-induced damage.

Xeroderma Pigmentosum: A Rapid Sensitive Method for Prenatal Diagnosis

When normal human cells, capable of repairing ultraviolet-induced lesions in their DNA, are incubated in the thymidine analog 5-bromodeoxyuridine after ultraviolet irradiation, the analog is incorporated into the repaired regions. When such repaired cells are subsequently irradiated with 313-nanometer radiation and placed in alkali, breaks appear in the DNA at sites of incorporation of 5bromodeoxyuridine, inducing a dramatic downward shift in the sedimentation constant of the DNA. Cells from patients with the disease xeroderma pigmentosum, which causes sensitivity to ultraviolet, are incapable or only minimally capable of repair; such cells incorporate little 5-bromodeoxyuridine into their DNA under these conditions and, upon 313-nanometer irradiation and sedimentation in alkali, exhibit only minor shifts in DNA sedimentation constants. When fibroblasts developed from biopsies of normal skin and of skin from patients with xeroderma pigmentosum, as well as cells cultured from midtrimester amniotic fluid, were assayed in this fashion unequivocal differences between normal and xeroderma pigmentosum cells were shown. Xeroderma pigmentosum heterozygotes are clearly distinguishable from homozygous mutants, and results are available 12 hours after irradiation.

Recovery of the ability to synthesize DNA in segments of normal size at long times after ultraviolet irradiation of human cells.

DNA synthesized in human cells within the first hour after ultraviolet (UV) irradiation is made in segments of lower molecular weight than in nonirradiated cells. The size of these segments approximates the average distance between pyrimidine dimers in the parental DNA. This suggests that the dimers interrupt normal DNA synthesis and result in gaps in the newly synthesized DNA. However, DNA synthesized in human cells at long times after irradiation is made in segments equal or nearly equal to those synthesized by nonirradiated cells. The recovery of the ability to synthesize DNA in segments of normal size occurs in normal human cells, where the dimers are excised, and also in cells of the human mutants xeroderma pigmentosum (XP), where the dimers remain in the DNA. This observation implies that the pyrimidine dimer may not be the lesion that causes DNA to be synthesized in smaller than normal segments.

Mutagenicity screening of five methyl carbamate insecticides and their nitroso derivatives using mutants of Salmonella typhimurium LT2

The mutagenic activity of five methyl carbamate insecticides-carbaryl, baygon, BUX-Ten, landrin and methomyl-and their nitroso derivatives were investigated using histidine auxotrophs-his TA98, his TA100, his TA1535, his TA1537 and his TA1538--of Salmonella typhimurium LT2 derived by Ames. The methyl carbamate insecticides did not cause a signficant increase in the number of revertant colonies in any of the strains used. In contrast, the nitroso derivatives of the carbamate insecticides greatly increased the number of colonies on plates inoculated with strains his TA100 and his TA1535. We conclude that the nitroso derivatives of the tested methyl carbamate insecticides are potent mutagens; whereas, the parent insecticides are non-mutagenic.

Effect of Caffeine on Postreplication Repair in Human Cells

DNA synthesized shortly after ultraviolet (UV) irradiation of human cells is made in segments that are smaller than normal, but at long times after irradiation the segments made are normal in size. Upon incubation, both the shorter and the normal segments are elongated and joined by the insertion of exogenous nucleotides to form high molecular weight DNA as in nonirradiated cells. These processes occur in normal human cells, where UV-induced pyrimidine dimers are excised, as well as in xeroderma pigmentosum (XP) cells, where dimers are not excised. The effect of caffeine on these processes was determined for both normal human and XP cells. Caffeine, which binds to denatured regions of DNA, inhibited DNA chain elongation and joining in irradiated XP cells but not in irradiated normal human or nonirradiated cells. Caffeine also caused an alteration in the ability to recover synthesis of DNA of normal size at long times after irradiation in XP cells but not in normal cells.

Defective repair of gamma-ray induced DNA damage in xeroderma pigmentosum cells.

We used the bromouracil-photolysis technique to estimate the sizes of the repaired regions in normal human and xeroderma pigmentosum (XP) cells irradiated by gamma-rays aerobically or anoxically. After 1 1/2 hours of incubation, single-strand breaks were repaired and the repaired regions were small--one to two BrUra residues--for cells irradiated aerobically or anoxically. After a 20-hour incubation, the repaired region in normal cells showed a component mimicking U.V.-repair. There were large patches (approximately 30 BrUra residues) in the approximate ratios of one per six chain breaks for aerobic irradiation and one per three chain breaks for anoxic irradiation. XP cells, however, only showed large patches at 20 hours if they had been irradiated aerobically. We could not detect such regions in XP cells irradiated anoxically. These results indicate (1) that some part of ionizing damage mimics excision of U.V. damage in that the repair patches are large and the repair takes an appreciable time; (2) the types of such damage depend on whether the irradiation is done aerobically or anoxically; and (3) XP cells are defective in repairing a component of anoxic damage.