A STUDY OF THE RELATIONSHIP BETWEEN SURVIVAL AND REPAIR SYNTHESIS'' OF DNA AFTER ULTRAVIOLET LIGHT EXPOSURE

Abstract— The influence of amino acid prestarvation on both the resistance to u.v. and the postirradiation repair synthesis of E. coli 15 T^- 555-7 thy meth arg trp and E. coli B/r (HCR⁺) was followed. Prestarvation increased the number of survivors about 30–100 fold in both strains at doses 600-1200ergs/mm². In contrast to survival no increase in repair synthesis was observed. Thus, the increase in survival has to be brought about by a mechanism which seems to be independent of additional repair synthesis.     

RECOVERY OF SUBCHROMOSOMAL DNA SYNTHESIS IN SYNCHRONOUS V-79 CHINESE HAMSTER CELLS AFTER ULTRAVIOLET LIGHT EXPOSURE

Abstract— Previous work obtained from Chinese hamster V-79 cells indicated that, immediately following exposure, UV-induced lesions acted as blocks to elongation of nascent strands, but gradually lost that ability over a 10 h period after exposure to 10 J/m². The work reported herein attempted to examine possible cell cycle mediated alterations in the recovery of DNA synthesis. Kinetic incorporation of radiolabeled thymidine studies indicated that there may have been a more rapid recovery of DNA synthesis in cells irradiated in G₁ or G₂ vs cells irradiated in S phase. DNA fiber autoradiograms prepared from synchronous cells indicated that after irradiation in any phase of the cell cycle, the length of newly synthesized DNA was equal to control lengths 1 h after exposure to 5.0 J/m² (or 1 h after entering S phase for cells irradiated in G₁ or G₂). This observed recovery was not solely due to an excision process. No cell cycle mediated difference in the number of dimers induced or removed as a function of cell cycle position was observed. These results appear to be consistent with a continuum of effects, with initiation effects dominating the response at low fluences, gapped synthesis at intermediate fluences and elongation inhibition at high fluences. The fluences at which each event dominates may be cell-line specific.     

DNA SYNTHESIS-KINETICS, CELL DIVISION DELAY, AND POST-REPLICATION REPAIR AFTER UV IRRADIATION OF FROZEN CELLS OF E. COLI B/r

Abstract— Previous studies have shown that the relative yields of photoproducts produced in the DNA of Escherichia coli cells UV irradiated at -79°C differ from those produced at +21°C; the yield of DNA-protein cross-links was markedly enhanced at -79°C while the yield of thymine dimers was reduced. In the present studies, cells of E. coli B/r thy were frozen at -79°C, and then UV irradiated (254nm) while frozen(4.7 J m^-2), or after thawing (22 Jm^-2). Essentially the same survival, cell division delay, and DNA synthesis kinetics were observed for these two samples after irradiation, even though the UV fluence differed by a factor of ˜5. This supports previous observations that a correlation exists between the magnitude of the effects of UV radiation upon DNA synthesis kinetics and on cell survival. The weight average molecular weight of the pulse labeled DNA in the sample irradiated at +21°C was one-half that of the sample irradiated at -79°C, and complete repair of daughter-strand gaps was observed in both cases. Thus, UV-induced lesions produced in cells at -79°C (i.e. DNA-protein cross-links) appear to be amenable to post-replicational repair. While the overall DNA synthesis kinetics were the same for the two irradiation procedures, the apparent number of lesions produced per unit length of DNA was not. This suggests that each of the lesions produced in frozen cells, although apparently fewer in number, must cause a longer local delay in DNA synthesis than those lesions produced at +21°C.     

THE EFFECTS OF ULTRAVIOLET LIGHT ON HOST CELL REACTIVATION AND PLAQUE SIZE OF Herpes simplex VIRUS TYPE I IN C3H/lOT1/2 MOUSE CELLS

— Herpes simplex virus — type 1 (HSV-I) plaque-forming ability and plaque size were measured on C3H/1OT1/2 cell monolayers as functions of pretreatment dose with UV light at different times before inoculation with virus, in order to determine if UV-enhanced reactivation (ER) of UV-irradiated virus. as well as associated phenomena, could be obtained in this cell system. The number of virus plaques observed (i.e. the capacity of the cells to support virus growth) and the size of the plaques were found to increase substantially with pretreatment of the cells with UV light. However, no significant ER was observed. Therefore, the mechanisms responsible for the increases in plaque size and cell capacity seem to be independent of those responsible for ER. In work by others. C3H/l0T1/2 cells have hcen transformed by UV light at doses similar to those used in this study; the absence of ER of UV-irradiated virus in this study indicates that the mechanism underlying ER is not required for transformation.     

CELL DENSITY DEPENDENCE OF ULTRAVIOLET LIGHT ENHANCED REACTIVATION OF Herpes simplex TYPE I AND THE LARGE PLAQUE EFFECT IN C3H/10T1/2 MOUSE FIBROBLASTS

C3H/10T1/2 mouse fibroblasts were grown to different cell densities either by plating at low density and allowing different growth periods, or by plating at a series of increasing densities and allowing the same growth period. These plates were UV irradiated at 7.5 J/m2 or mock irradiated and 24 h later infected with UV-irradiated Herpes simplex type I virus which had been UV irradiated at 50 or 125 J/m2 or mock irradiated. The numbers and sizes of plaques were measured and these data used to calculate the extent of UV-enhanced host cell reactivation, the capacity enhancement, the large plaque effect (LPE) and the small plaque effect (SME). The influence of cell density on these phenomena was similar for both series of density experiments. Ultraviolet-enhanced host cell reactivation could be demonstrated only for cultures of lower density. The capacity of the cells for Herpes simplex type I virus decreased with cell density, but UV irradiated cells showed an increase in capacity with cell density. Plaque sizes decreased in all cases with cell density but the LPE and SPE were not significantly altered. The greatest variation in the above parameters occurred just as the cells were approaching confluence, where most host cell reactivation experiments are carried out. We conclude that the reproducibility of such experiments depends critically on cell density, a dependence which may be relevant to mechanistic interpretations of the UV-dependent phenomena.     

DIFFERENT EFFECTS OF RAD GENES OF SACCHAROMYCES CEREVISIAE ON INCISIONS OF INTERSTRAND CROSSLINKS AND MONOADDUCTS IN DNA INDUCED BY PSORALEN PLUS NEAR UV LIGHT TREATMENT

Abstract— The RAD1, RAD2, RAD3 and RAD4 genes of Saccharomyces cerevisiae are required for incising DNA containing UV induced pyrimidine dimers or psoralen plus 360 nm light induced interstrand crosslinks. We have now determined if these genes are also required for incising DNA at psoralen plus 360 nm light induced monoadducts. For distinguishing between incision breaks occurring at crosslinks and at monoadducts. we have used the cdc9-2 mutant, defective in DNA ligase activity at the restrictive temperature, and the radl-2 cdc9-2, rad2-5 cdc9-2 , rad3-2 cdc9-2 and rad4-4 cdc9-2 double mutant combinations. We conclude that the radl, rad2 , and rad4 mutants are defective in incising DNA both at crosslinks and monoadducts, whereas the rad3 mutant is proficient in incising DNA at monoadducts but not at crosslinks.     

EVALUATION OF DNA CROSSLINKS AND MONOADDUCTS IN MOUSE EMBRYO FIBROBLASTS AFTER TREATMENT WITH MONO-AND BIFUNCTIONAL FUROCOUMARINS AND 365 nm (UVA) IRRADIATION. POSSIBLE RELATIONSHIP TO CARCINOGENICITY

Abstract— Formation of crosslinks in DNA by various mono- and bifunctional furocoumarins plus UVA light in mouse embryo fibroblasts was evaluated by a NaI density gradient centrifugation method. Angelicin and 3-carbethoxypsoralen did not form any crosslinks: however, angelicin was slightly carcinogenic for mouse skin, whereas 3-carbethoxypsoralen was not carcinogenic. Psoralen induced DNA crosslinking, in a dose-dependent fashion and was highly carcinogenic for mouse skin. In contrast to the psoralen-induced photoadducts (120 per 10⁶ nucleotides) which were left unrepaired, 41% of the 3-carbethoxypsoralen adducts (30 per 10⁶ nucleotides) were removed during 1 h of dark post-incubation.     

DNA SINGLE-STRAND LESIONS DUE TO ''SUNLIGHT'' AND UV LIGHT: A COMPARISON OF THEIR INDUCTION IN CHINESE HAMSTER AND HUMAN CELLS, AND THEIR FATE IN CHINESE HAMSTER CELLS

Abstract— It has been shown that the lethal properties of germicidal UV light (254 nm) and sunlight-simulating near UV light are qualitatively different (Elkind et al ., 1978). Further to compare these two radiations, the induction of single-strand DNA breaks (i.e. frank breaks plus alkali-labile lesions) was measured in two cell lines. Equal numbers of breaks in Chinese hamster cells require a dose of UV 5.5% of a near UV dose but in HeLa cells a UV dose of 7.6% of a near-UV dose is required. The rate of break production by these radiations is about 1/10-th of that due to X-rays when a comparison is made on an equal killing dose basis. The inventory of breaks in Chinese hamster cells was also followed and was found to be characteristically different for UV compared to near UV light. These data indicate that significant differences exist, at a molecular level, in the effects produced by ultraviolet and sunlight-simulating light, and further emphasize the need for caution in attempting to extrapolate from observed molecular or biological effects due to the former to those to be expected from the latter.