The responses of Escherichia coli to X rays and hydrogen peroxide were examined in mutants which are deficient in one or more DNA repair genes. Mutant cells deficient in either exonuclease III (xthA) or endonuclease IV (nfo) had normal resistance to X rays, but an xthA-nfo double mutant showed a sensitivity increased over that of either parental strain. A DNA polymerase I mutant (polA) was more sensitive than the xthA-nfo mutant. Cells bearing mutations in all of the polA, xthA, and nfo genes were more sensitive to X rays than polA and xthA-nfo mutants. Similar repair responses were obtained by exposing these mutant cells to hydrogen peroxide, with the exception of the xthA mutant, which was hypersensitive to this agent. The DNA polymerase III mutant (polC(Ts)) was slightly more sensitive to the agents than the wild-type strain at the restrictive temperature. The sensitivity of the polC-xthA-nfo mutant to X rays and hydrogen peroxide was greater than that of polC but almost the same as that of the xthA-nfo mutant. From these results it appears that there are at least four repair pathways, the DNA polymerase I-, exonuclease III/endonuclease IV and DNA polymerase I-, exonuclease III/endonuclease IV and DNA polymerase III-, and exonuclease III/endonuclease IV-dependent pathways, for the repair of oxidative DNA damages in E. coli.

The effect of X irradiation on the major proteins of human erythrocyte membranes was investigated with respect to a difference in radiosensitivity. The electrophoretic patterns on sodium dodecyl sulfate/polyacrylamide gels revealed that, among seven major proteins, band 1 and 2 proteins, both called spectrin, preferentially disappeared on the gels following X irradiation (100-400 Gy). Spectrin was at least partly transformed into mutually cross-linked aggregates which did not enter the polyacrylamide gels. The most significant effect of the action of X rays was the radiolytic fragmentation of spectrin to short polypeptide pieces having smaller molecular weights than the normal spectrin monomers. These effects of X irradiation could also be detected with purified spectrin molecules. The other major protein bands, 3 to 7, did not diminish to a notable extent. It was also observed that X rays induce changes in the shape of isolated ghosts with a dose-response relationship similar to that for the loss of bands 1 and 2 on the gels. It is suggested that the shape changes of ghosts might be the consequence of radiation-induced structural alterations of spectrin.

The changes in the fluidity of erythrocyte membranes following X irradiation were investigated with the aid of a fluorescent aromatic hydrocarbon, pyrene. The method used in the present experiments, pyrene excimer fluorescence, is based upon the microviscosity dependence of the lateral diffusion rate of the probe in the membrane plane. The lateral diffusion rate obtained by this fluorescence technique can provide valuable information about the fluidity of the membrane environment of the probe. It was found that the excimer/monomer ratio, a parameter of the lateral diffusion rate, of pyrene bound to erythrocyte membranes irradiated with X rays was higher than that in unirradiated membranes. This effect was observable when 100 R were given to the membranes. In addition, the fluorescence polarization of membrane-bound pyrene decreased in X-irradiated membranes. From these results it was concluded that X irradiation can induce an increase in the fluidity of the hydrocarbon regions of the membranes.

The structural changes in erythrocyte membranes caused by X irradiation were investigated with the aid of fluorescent probes. It was found that the fluorescence intensity of 1-anilino-8-naphthalene sulfonate (ANS) combined with X-irradiated membranes decreased as a function of the dose. The effect could be observed even when 100 R was given. The lowering of the quantum yield of ANS fluorescence in the irradiated membranes seemed to be the primary cause of the fluorescence decrease, as the amount of bound ANS was found to be only slightly decreased. The fluorescence decrease was not accompanied by a significant shift of the emission spectrum to a longer wavelength. In addition, the fluorescence polarization of bound ANS decreased in X-irradiated membranes, suggesting that the decrease in quantum yield of bound ANS might be due to the decrease in membrane microviscosity. Measurement of the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene incorporated into the membranes indicated that it also decreased with increasing X-ray doses. The changes in the dynamic properties of erythrocyte membranes caused by X irradiation were further confirmed. From these results it was concluded that X irradiation readily induces significant changes of erythrocyte membrane structure.