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Purpose: To determine whether yeast cells (Saccharomyces cerevisiae) defective in damage-inducible cell-cycle arrest can invoke an adaptive response and become resistant to normally lethal doses of ionizing radiation., Materials and Methods: Wild-type yeast cells, cells defective for DNA-damage-responsive G1 and G2 cell-cycle arrest (rad9delta), and cells defective for recombinational repair of DNA damage (rad50, 51, 52) were subjected to adapting treatments of heat or radiation and subsequently exposed to normally lethal doses of radiation. Survival, as measured by colony-forming ability, was compared with non-adapted, control cells., Results: Wild-type and rad9delta cells became more resistant to potentially lethal doses of radiation after exposure to conditions that are known to elicit the adaptive response. Further, the relative magnitude of resistance developed by the normal, wild-type and rad9delta yeast cells was similar, with a dose modifying factor (at D1) for radiation-induced radiation resistance of 1.3 for both strains. Dose modifying factors (at D1) for heat-induced radiation resistance were 1.7 and 1.6 for wild-type and rad9delta cells, respectively. In contrast, none of the recombinational repair-defective cells exhibited radiation resistance after an adapting treatment., Conclusions: The ability of yeast cells to arrest in cell-cycle gap phases did not appear to contribute significantly to radiation resistance induced by radiation or heat. Instead, it is suggested that the adaptive response was due mainly to the existence and enhancement of cellular recombinational repair capacity, which was sufficient to repair any DNA damage without the requirement of a detectable cell-cycle delay.

Cis-diamminedichloroplatinum II (cisplatin) is a DNA inter- and intrastrand crosslinking agent which can sensitize prokaryotic and eukaryotic cells to killing by ionizing radiation. The mechanism of radiosensitization is unknown but may involve cisplatin inhibition of repair of DNA damage caused by radiation. Repair proficient wild type and repair deficient (rad52, recombinational repair or rad3, excision repair) strains of the yeast Saccharomyces cerevisiae were used to determine whether defects in DNA repair mechanisms would modify the radiosensitizing effect of cisplatin. We report that cisplatin exposure could sensitize yeast cells with a competent recombinational repair mechanism (wild type or rad3), but could not sensitize cells defective in recombinational repair (rad52), indicating that the radiosensitizing effect of cisplatin was due to inhibition of DNA repair processes involving error free RAD52-dependent recombinational repair. The presence or absence of oxygen during irradiation did not alter this radiosensitization. Consistent with this result, cisplatin did not sensitize cells to mutation that results from lesion processing by an error prone DNA repair system. However, under certain circumstances, cisplatin exposure did not cause radiosensitization to killing by radiation in repair competent wild type cells. Within 2 h after a sublethal cisplatin treatment, wild type yeast cells became both thermally tolerant and radiation resistant. Cisplatin pretreatment also suppressed mutations caused by exposure to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a response previously shown in wild type yeast cells following radiation pretreatment. Like radiation, the cisplatin-induced stress response did not confer radiation resistance or suppress MNNG mutations in a recombinational repair deficient mutant (rad52), although thermal tolerance was still induced. These results support the idea that cisplatin adducts in DNA interfere with RAD52-dependent recombinational repair and thereby sensitize cells to killing by radiation. However, the lesions can subsequently induce a general stress response, part of which is induction of RAD52-dependent error free recombinational repair. This stress response confers radiation resistance, thermal tolerance, and mutation resistance in yeast.

We have investigated the role of heat-shock protein 104 (hsp-104) in the induction of ionizing radiation resistance in yeast. Yeast defective in the production of hsp-104 are constitutively sensitive to killing by heat and are known to be compromised for the induction of thermotolerance. We confirmed that hsp104 yeast mutants are altered in their ability to become thermotolerant by a heat stress. However, we found that the mutant strain did respond to heat shock and increased its resistance to a second lethal heat stress, although the magnitude of the increase was much lower than for the parental wild-type strain. Here we report that the constitutive level of ionizing radiation resistance was higher in the hsp104 mutant compared to wild-type cells and that heat shock induced increased radioresistance in both strains. When radiation was used as the stressing agent, thermal resistance was fully induced in wild-type cells and only slightly induced in the hsp104 mutant. In comparison, radioresistance was induced in both cell strains by a radiation stress. The increased radiation resistance was always slightly higher in the hsp104 mutant but developed with similar kinetics in both cell strains. Entry into stationary growth phase is another event known to induce both thermal tolerance and radiation resistance in yeast. In these experiments, entry into stationary phase induced similar levels of thermal tolerance in the two strains but higher levels of radioresistance in hsp104 mutants. The activation of both resistance mechanisms occurred earlier in the hsp104 mutants compared to wild-type cells. These results show that hsp-104 is not essential for either the process by which the cell recognizes and responds to any of these stresses, or the mechanisms by which the cell actually elevates its resistance to heat and radiation. We conclude, however, that hsp-104 is important in regulating the development and magnitude of induced radiation resistance as well as induced thermal tolerance in yeast.

Saccharomyces cerevisiae and Chlamydomonas reinhardtii respond to a sublethal exposure of ionizing radiation by increasing their resistance to killing by a second exposure. We demonstrate here that the two lower eukaryotes apparently achieve this by different mechanisms. We have shown that induced radioresistance in yeast results from increased capacity for recombinational repair, which we believe to occur in G2-phase haploid cells by recombination between homologous chromosomes. This is not possible in G1-phase haploid cells, which lack a second copy of DNA. Haploid C. reinhardtii cells, however, show induced resistance when irradiated asynchronously or in the G1 phase of the cell cycle. We have shown previously that the development of radiation resistance in yeast is proportional to the magnitude of the inducing dose and clearly demonstrates an oxygen effect. There was no oxygen effect for induced radiation resistance in C. reinhardtii cells, but induction remained proportional to dose. In yeast we have reported that both increased radioresistance and thermotolerance are inducible by a heat shock. Here, C. reinhardtii showed induced thermotolerance but no induced radioresistance in response to a heat stress. We have also determined previously that the induced recombinational DNA repair system in yeast recognizes alkylation lesions and therefore confers increased resistance to mutation by MNNG. In these experiments, C. reinhardtii induced for radioresistance were not more resistant to MNNG mutagenesis. These data indicate that haploid C. reinhardtii has a unique DSB repair mechanism. We propose that one possible mechanism may involve chloroplast DNA in a cooperative chloroplast/nuclear recombinational repair process.

DNA recombinational repair, and an increase in its capacity induced by DNA damage, is believed to be the major mechanism that confers resistance to killing by ionizing radiation in yeast. We have examined the nature of the DNA lesions generated by ionizing radiation that induce this mechanism, using two different end points: resistance to cell killing and ability of the error-free recombinational repair system to compete for other DNA lesions and thereby suppress chemical mutation. Under the various conditions examined in this study, the "maximum" inducible radiation resistance was increased approximately 1.5- to 3-fold and suppression of mutation about 10-fold. DNA lesions produced by low-LET gamma rays at doses greater than about 20 Gy given in oxygen were shown to be more efficient, per unit dose, at inducing radioresistance to killing than were lesions produced by neutrons (high-LET radiation). This suggests that DNA single-strand breaks are more important lesions in the induction of radioresistance than DNA double-strand breaks. Oxygen-modified lesions produced by gamma rays (low-LET radiation) were particularly efficient as induction signals. DNA damage due to hydroxyl radicals (OH.) derived from the radiolytic decomposition of H2O produced lesions that strongly induced this DNA repair mechanism. Similarly, OH. derived from aqueous electrons (e-aq) in the presence of N2O also efficiently induced the response. Cells induced to radioresistance to killing with high-LET radiation did not suppress N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-generated mutations as well as cells induced with low-LET radiation, supporting the conclusion that the type of DNA damage produced by low-LET radiation is a better inducer of recombinational repair. Surprisingly, however, cells induced with gamma radiation in the presence of N2O that became radioresistant to killing were unable to suppress MNNG mutations. This result indicates that OH. generated via e-aq (in N2O) may produce unusual DNA lesions which retard normal repair and render the system unavailable to compete for MNNG-generated lesions. We suggest that the repairability of these unique lesions is restricted by either their chemical nature or topological accessibility. Attempted repair of these lesions has lethal consequences and accounts for N2O radiosensitization of repair-competent but not incompetent cells. We conclude that induction of radioresistance in yeast by ionizing radiation responds variably to different DNA lesions, and these affect the availability of the induced recombinational repair system to deal with subsequent damage.