Genetic control of mitotic crossing-over in yeasts. III. Induction by 8-methoxypsoralen and long-wave UV irradiation (lambda=365 nm)

The lethal effect of 8-methoxypsoralen (8-MOP) plus 365 nm light has been studied in haploid radiosensitive strains of Saccharomyces cerevisiae. The diploid of wild type and the diploid homozygous for the rad2 mutation (this mutation blocks the excision of UV-induced pyrimidine dimers) were more resistant to the lethal effect of 8-MOP plus 365 nm light than the haploid of wild type and rad2 haploid, respectively. The diploid homozygous for rad54 mutation (the mutation blocks the repair of double-strand breaks in DNA) was more sensitive than haploid rad54. The method of repeated irradiation allowed to study the capacity of radiosensitive diploids to remove monoadducts induced by 8-MOP in DNA. This process was very effective in diploids of wild type and in the rad54 rad54 diploid, while the rad2 rad2 diploid was characterized by nearly complete absence of monoadduct excision. The study of mitotic crossing over and mitotic segregation in yeast diploids, containing a pair of complementing alleles of the ade2 gene (red/pink) has shown a very high recombinogenic effect of 8-MOP plus 365 nm light. The rad2 mutation slightly increased the frequency of mitotic segregation and mitotic crossing over. The rad54 mutation decreased the frequency of mitotic segregation and entirely suppressed mitotic crossing over. The method of repeated irradiation showed that the cross-links, but not monoadducts, are the main cause of high recombinogenic effect of 8-MOP plus 365 nm light. The possible participation of different repair systems in recombinational processes induced by 8-MOP in yeast cells is discussed.     

8-Methoxypsoralen plus 365 nm light effects and repair in yeast.

Haploid wild-type and mutant cells of Saccharomyces carrying one of the single genes rad2-20 or rad9-4 and the double mutant rad2-20rad9-4 were tested for their response to a treatment with 8-methoxypsoralen plus 365 nm light using immediate and delayed plating techniques. The mutant defective in the excision of ultraviolet-induced pyrimidine dimers (rad2-20) as well as that presumably deficient in a recombinational repair system (rad9-4) are more sensitive than wild type cells. The double mutant (rad2-20rad9-4) demonstrates a higher sensitivity than each of the single mutants, indicating that at least two pathways are involved in the repair of the 8-methoxypsoralen plus 365 nm induced damages. In all cases survival curves have shoulders. The survival of wild type and rad9-4 cells is increased after dark holding whereas it remains constant for the rad2-20 mutant and for the double mutant. These results show that the induced damages are reparable. Respiratory deficient mutant (p-) were compared to the corresponding respiratory competent cells. It is shown that the respiratory function is required for the expression of the excision repair activity. The 8-methoxypsoralen plus 365 nm ligh treatment appears to be less effective than ultraviolet irradiation (254 nm) in the induction of the cytoplasmic 'petite' mutation at the same survival levels.     

Use of 8-methoxypsoralen and light (lambda--365 nm) for studying repair in yeasts]

The lethal effect of 8-metoxypsoralen (8/MOP) plus light (lambda = 365 nm) on the haploid radioresistant and UV-sensitive strains of Saccharomyces cerevisiae was studied. The mutation uvs1 increased the sensitivity to the lethal effect of 8-MOP more than 2.8 times as compared to radioresistant strain. The method of repeated irradiation allowed to study kinetics of excision of monoadducts induced by 8-MOP. The mutant uvs1 was characterized by the absence of excision of monoadducts. The radioresistant strain removed monoadducts very efficiently (80%) after the incubation in complete liquid medium for 2.5 hours at 28 degrees before repeated irradiation. After the incubation of this strain in buffer (pH 7.0) monoadducts were removed considerably less efficiently (30%).     

Dose-rate effects of 8-methoxypsoralen plus 365-NM irradiation on cell killing in Saccharomyces cerevisiae.

Tow types of dose-rate effect that alter the survival response of haploid yeast cells to 8-methoxypsoralen (8-MOP) plus treatment with irradiation at 365 nm were studied. (1) When the concentration of 8-MOP was varied between 9.2 X 10(-5) and 2.3 X 10(-8) M and the dose rate of 365-nm irradiation kept constant, the efficiency of the irradiation for killing increased relatively to that of 8-MOP whe the concentration of 8-MOP decreased. This indicated that there was no strict reciprocity between radiation dose and concentration of drug. (2) When the dose rate of radiation was varied between 0.66 X 10(3) and 108 X 10(3) J m-2 h-1 and the concentration of 8-MOP was kept constant, the survival of wild-type cells increased strikingly at low dose rates of radiation as compared with high dose rates. Cells responded more to changes at low dose rates than to equal changes a high dose rates. The high resistance of wild-type cells to 8-MOP plus radiation delivered at low dose rates absent from rad 1-3 cells defective in excision-repair. This suggests that the dose-rate effect seen in wild-type cells depended at least in part on an active excision-repair function. At low dose rates of radiation, the shoulder of the survival curve for rad1-3 cells, i.e. the ability to accumulate sub-lethal damage, was increased by a factor of about 2 when compared with that seen at a high dose rate. Thus it is likely that at low dose rates a repair function other than excision-resynthesis may operate in rad1-3 cells.     

Repair of plasmid DNA treated with 8-methoxypsoralen and long-wave UV light (lambda=365 nm) in wild type and mutant rad2 cells of Saccharomyces cerevisiae

The method of repeated irradiation has been used to study excision of 8-MOP monoadducts from plasmid and chromosomal DNA in cells of wild type and rad2 mutant of Saccharomyces cerevisiae. The measurement of kinetics of monoadduct removal from chromosomal DNA in intact and competent yeast cells showed that monoadducts were excised in both types of cells with normal repair, but this process was blocked in intact and competent cells of the rad2 mutant. The survival of pYF91 plasmid treated in vitro with 8-MOP plus near UV-light has been studied in the cells of the wild type and in incision-defective rad2 mutant by the measurement of cell transformation frequency. Episomic pYF91 plasmid used in these experiments contained the yeast nuclear LEU2 gene, a portion of 2 mkm DNA and DNA of bacterial plasmid pBR322 with resistance to ampicillin. The pYF91 plasmid was treated with 8-MOP plus near UV-light in vitro, then unbound 8-MOP was removed by dialysis. This DNA was used for transformation. The transformed yeast cells were irradiated repeatedly. The quantitative alteration of the yield of transformants, depending on the time of keeping these yeast cells in complete liquid medium at 30 degrees C, prior to repeated irradiation, allowed to measure the kinetics of monoadduct excision from plasmid DNA. It was shown that monoadducts were removed equally effectively from plasmid DNA introduced into cells of the wild type and rad2 mutant. Possibly, the repair system of both these strains provides excision of monoadducts from plasmid DNA, but this process is blocked in the rad2 mutant, relatively to monoadduct excision from chromosomal DNA.     

Intragenic mitotic recombination induced by ultraviolet and gamma rays in radiosensitive mutants of Saccharomyces cerevisiae yeasts

The effect of UV- and gamma-irradiation on the survival and intragenic mitotic recombination (gene conversion) of 5 radiosensitive mutants was studied in comparison with the wild type. The level of spontaneous conversion was similar for RAD, rad2 and rad15, mutations xrs2 and xrs4 increasing and rad54 significantly decreasing it. The frequency of conversion induced by UV-light was greater in rad2, rad15 and xrs2 mutants and lower in xrs4, as compared to RAD. Gamma-irradiation caused induction of gene conversion with an equal frequency in RAD, rad2, rad15. Xrs2 and xrs4 mutations slightly decreased gamma-induced conversion. In rad54 mutant, UV-and gamma-induced conversion was practically absent. In the wild type yeast, a diploid strain is more resistant than a haploid, whereas in rad54 a diploid strain has the same or an increased sensitivity, as compared to a haploid strain (the "inverse ploidy effect"). This effect and also the block of induced mitotic recombination caused by rad54 indicate the presence in the yeast Saccharomyces cerevisiae of repair pathways of UV- and gamma-induced damages acting in diploid cells and realised by recombination. The data obtained as a result of many years' investigation of genetic effects in radiosensitive mutants of yeast are summarised and considered.     

Incision and postincision steps of pyrimidine dimer removal in excision-defective mutants of Saccharomyces cerevisiae.

cdc9, a temperature-sensitive mutant defective in polynucleotide deoxyribonucleic acid (DNA) ligase activity, accumulates low-molecular-weight DNA fragments (as measured by sedimentation of DNA in alkaline sucrose gradients) at the nonpermissive temperature after irradiation with ultraviolet light. This phenotype of cdc9 is a sensitive indicator of successful incision during excision repair of dimers. In strains containing excision-defective mutations in any of nine genes in combination with the cdc9 mutation, the absence of low-molecular-weight DNA at the nonpermissive temperature after ultraviolet treatment suggests that these mutants are incision defective, whereas the presence of low-molecular-weight DNA indicates that the mutants are defective in a step after incision. With rad1, rad2, rad3, rad4, and rad10 mutants, the molecular weight of the DNA remained unchanged after ultraviolet irradiation and incubation at the restrictive temperature, despite the presence of the cdc9 mutation; these mutants are therefore incision defective. Low-molecular-weight DNA was observed in rad14 cdc9 and rad16 cdc9 strains. With the rad16 strain, the accumulation of low-molecular-weight DNA correlated with the amount of excision taking place, whereas in the rad14 mutant strain, no evidence of dimer removal was obtained. Therefore, rad14 is likely to be defective in a step after incision.     

Genetic control of excision of Saccharomyces cerevisiae interstrand DNA cross-links induced by psoralen plus near-UV light.

Excision of interstrand DNA cross-links induced by 4,5',8-trimethyl psoralen plus 360-nm light was examined in wild type (RAD+) and various radiation-sensitive (rad) mutants of Saccharomyces cerevisiae known to be defective in the excision of UV light-induced pyrimidine dimers. Alkaline sucrose sedimentation of DNA after incubation of psoralen-plus-light-treated cells indicated little or no nicking of cross-linked DNA in rad1-2, rad2-5, rad3-2, rad4-4, rad10-2, and mms19-1 mutants. In the rad14-2 mutant, substantial nicking was observed but to a much lesser extent than in the RAD+ strains, whereas the rad16-1 mutant was as proficient in nicking as the RAD+ strain. Removal of cross-links was also examined in RAD+, rad3-2, and rad14-2 strains by determining the sensitivity of alkali-denatured and -neutralized DNA to hydrolysis by S1 nuclease. No cross-link removal was observed in the rad3-2 mutants, and the rad14-2 mutant was much less efficient than the RAD+ strain in removing cross-links.     

Fate of photo-induced 8-methoxypsoralen mono-adducts in yeast. Evidence for bypass of these lesions in the absence of excision repair

A fraction of UVA-induced 8-methoxypsoralen (8-MOP) mono-adducts can be transformed by a second UVA (365 nm) irradiation procedure into lethal cross-links in Saccharomyces cerevisiae. To follow the fate of cross-linkable mono-adducts, cells were incubated in complete medium between the two UVA doses and survival was measured. The killing effect of the second UVA dose decreases rapidly in haploid wild-type as well as in strains blocked in mutagenic (RAD6+ type) or in recombinogenic (RAD52+ type) repair pathways. This is also true in the pso1-1 and pso2-1 strains selected for sensitivity to 8-MOP plus UVA treatment. In contrast, persistence of mono-adducts is observed in strains blocked in the excision-resynthesis repair pathway. In other words, cross-linkable mono-adducts are repaired by the excision process. The use of the cell-cycle conditional mutant strain (cdc14-1) permitted us to apply the second dose at a specific cell-cycle stage (post-G2 phase) after a 'priming' UVA treatment on stationary (G1) phase cells. Such experiments showed a bypass of mono-adducts in an excision-deficient context for at least one round of DNA replication.     

Mutants Showing Heterothallism from a Homothallic Strain of SACCHAROMYCES CEREVISIAE

Reverse and forward mutation, induced by photoaddition of 8-methoxypsoralen (8-MOP) and 3-carbethoxypsoralen (3-CPs) or ultraviolet light (UV), are reduced in three pso mutants of Saccharomyces cerevisiae. The pso1–1 strain exhibits a lower frequency of spontaneous reversion (antimutator) and is almost entirely unaffected by the three agents in both the haploid and diploid states. The pso2–1 strain demonstrates very reduced frequencies of 8-MOP and 3-CPs plus 365 nm radiation-induced mutations in happloid and diploid cells. UV-induced mutations are slightly reduced, whereas survival is almost normal. The pso3–1 strain is mutable by 8-MOP and 3-CPs photoaddition only in the low-dose range. After UV treatment, survival of pso3–1 is nearly normal, whereas the frequencies of induced mutants are diminished as compared to the normal PSO⁺. An analogue of adenine, 6-N-hydroxyaminopurine, is capable of inducing reversions in wild type, as well as in pso and rad6–1 mutant strains, indicating that this drug may act as a direct mutagen in yeast. The comparison of photoaddition of the bifunctional agent (8-MOP) to that of the monofunctional one (3-CPs) confirms that cross-links, as well as monoadditions, are mutagenic in S. cerevisiae. Repair, of the recombinational type, taking place in diploid cells or in haploid cells in G2 phase leads to higher survival, but appears to be error-free.     

Unrepaired Heteroduplex DNA in Saccharomyces Cerevisiae Is Decreased in Rad1 Rad52-Independent Recombination

A direct repeat recombination assay between SUP4 heteroalleles detects unrepaired heteroduplex DNA (hDNA) as sectored colonies. The frequency of unrepaired heteroduplex is dependent on the mismatch and is highest in a construct that generates C:C or G:G mispairs and lowest in one that generates T:G or C:A mispairs. In addition, unrepaired hDNA increases for all mismatches tested in pms1 mismatch repair-deficient strains. These results support the notion that hDNA is formed across the SUP4 repeats during the recombination event and is then subject to mismatch repair. The effects of various repair and recombination defective mutations on this assay were examined. Unrepaired heteroduplex increases significantly only in rad52 mutant strains. In addition, direct repeat recombination is reduced 2-fold in rad52 mutant strains, while in rad51, rad54, rad55 and rad57 mutants direct repeat recombination is increased 3-4-fold. Mutations in the excision repair gene, RAD1, do not affect the frequency of direct repeat recombination. However, the level of unrepaired heteroduplex is slightly decreased in rad1 mutant strains. Similar to previous studies, rad1 rad52 double mutants show a synergistic reduction in direct repeat recombination (35-fold). Interestingly, unrepaired heteroduplex is reduced 4-fold in the double mutants. Experiments with shortened repeats suggest that the reduction in unrepaired heteroduplex is due to decreased hDNA tract length in the double mutant strain.     

S. cerevisiae has three pathways for DNA interstrand crosslink repair.

Yeast mutants, snm1 (pso2-1), rev3 (pso1-1), and rad51, which display significant sensitivity to interstrand crosslinks (ICLs) have low relative sensitivity to other DNA damaging agents. SNM1, REV3, and RAD51 were disrupted in the same haploid strain, singly and in combination. The double mutants, snm1 Delta rev3 Delta, snm1 Delta rad51 Delta and rev3 Delta rad51 Delta were all more sensitive to ICLs than any of the single mutants, indicating that they are in separate epistasis groups for survival. A triple mutant displayed greater sensitivity to ICLs than any of the double mutants, with one ICL per genome being lethal. Therefore, Saccharomyces cerevisiae appears to have three separate ICL repair pathways, but no more. S-phase delay was not observed after ICL damage introduced by cisplatin (CDDP) or 8-methoxypsoralen (8-MOP) during the G1-phase, in any of the above mutants, or in an isogenic rad14 Delta mutant deficient in nucleotide excision repair. However, the psoralen analog angelicin (monoadduct damage) induced a significant S-phase delay in the rad14 Delta mutant. Thus, normal S-phase in the presence of ICLs does not seem to be due to rapid excision repair. The results also indicate that monoadduct formation by CDDP or 8-MOP at the doses used is not sufficient to delay S-phase in the rad14 Delta mutant. While the sensitivity of a rev3 Delta mutant indicates Pol zeta is needed for optimal ICL repair, isogenic cells deficient in Pol eta (rad30 Delta cells) were not significantly more sensitive to ICL agents than wild-type cells, and have no S-phase delay.     

Cytological characterization of Chinese hamster ovary X-ray-sensitive mutant cells, xrs 5 and xrs 6. II. Induction of sister-chromatid exchanges and chromosomal aberrations by X-rays and UV-irradiation and their modulation by inhibitors of poly(ADP-ribose) synthetase and alpha-polymerase

The cell killing and induction of sister-chromatid exchanges (SCEs) by X-rays and short-wave ultraviolet (UV) irradiation in combination with inhibitors of DNA repair, 3-aminobenzamide (3AB), cytosine arabinoside (ara-C) or aphidicolin (APC) were studied in wild-type CHO-K1 and two X-ray-sensitive mutants, xrs 5 and xrs 6 cells. The spontaneous frequency of SCEs was similar in the mutants and the wild-type CHO-K1 cells (8.4-10.3 SCEs/cell). Though X-rays are known to be poor inducers of SCEs, a dose-dependent increase in the frequency of SCEs in xrs 6 cells (doubling at 150 rad) was found in comparison to a small increase in xrs 5 and no increase in wild-type CHO-K1 cells. 3AB, an inhibitor of poly(ADP-ribose) synthetase increased the spontaneous frequency of SCEs in all the cell types. 3AB did not potentiate the X-ray-induced frequency of SCEs in any of the cell lines. Ara-C, an inhibitor of DNA polymerase alpha, increased the frequency of SCEs in all the cell lines. In combined treatment with X-rays, ara-C had no synergistic effect in xrs 5 and xrs 6 cells, but the frequency of SCEs increased in X-irradiated wild-type CHO-K1 cells post-treated with ara-C. For the induced frequency of SCEs, CHO-K1 cells treated with X-rays plus ara-C behaved like xrs 6 cells treated with X-rays alone, suggesting a possible defect in DNA base damage repair in xrs 6 cells, in addition to the known defective repair of DNA double-strand breaks (DSBs). Survival experiments revealed higher sensitivity of xrs 5 and xrs 6 mutant cells to the cell killing effect of X-rays in S-phase when compared to wild-type CHO-K1 cells. The mutants responded with lesser sensitivity to cell killing effect of ara-C and APC than CHO-K1 cells, the relative sensitivity to ara-C or APC being CHO-K1 greater than xrs 5 greater than xrs 6 cells. When X-irradiation was coupled with ara-C, the results obtained for survival were similar to those of the SCE test, i.e., unlike wild-type CHO-K1, no synergistic effect was observed in xrs 5 or xrs 6 cells. After UV-irradiation, the frequency of SCEs increased similarly in wild-type CHO-K1 and xrs 6 cells, but xrs 5 cells responded with lower frequency of SCEs.(ABSTRACT TRUNCATED AT 400 WORDS)     

Excision repair of nitrogen mustard-DNA adducts in Saccharomyces cerevisiae.

The bifunctional alkylating anticancer drug nitrogen mustard forms a variety of DNA lesions, including monoadducts and intrastrand and interstrand crosslinks. Although it is known that nucleotide excision repair (NER) is important in processing these adducts, the role of the other principal excision repair pathway, base excision repair (BER) is less well defined. Using isogenic Saccharomyces cerevisiae strains disrupted for a variety of NER and BER genes we have examined the relative importance of the two pathways in the repair of nitrogen mustard adducts. As expected, NER defective cells (rad4 and rad14 strains) are extremely sensitive to the drug. One of the BER mutants, a 3-methyladenine glycosylase defective (mag1) strain also shows significant hypersensitivity. Using a rad4/mag1 double mutant it is shown that the two excision repair pathways are epistatic to each other for nitrogen mustard sensitivity. Furthermore, both rad14 and mag1 disruptants show elevated levels of nitrogen mustard-induced forward mutation. Measurements of repair rates of nitrogen mustard N-alkylpurine adducts in the highly transcribed RPB2 gene demonstrate defects in the processing of mono-adducts in rad4, rad14 and mag1 strains. However, there are differences in the kinetics of adduct removal in the NER mutants compared to the mag1 strain. In the mag1 strain significant repair occurs within 1 h with evidence of enhanced repair on the transcribed strand. Adducts however accumulate at later times in this strain. In contrast, in the NER mutants repair is only evident at times greater than 1 h. In a mag1/rad4 double mutant damage accumulates with no evidence of repair. Comparison of the rates of repair in this gene with those in a different genomic region indicate that the contributions of NER and BER to the repair of nitrogen mustard adducts may not be the same genome wide.     

RAD50 and RAD51 define two pathways that collaborate to maintain telomeres in the absence of telomerase

Telomere length is maintained by the de novo addition of telomere repeats by telomerase, yet recombination can elongate telomeres in the absence of telomerase. When the yeast telomerase RNA component, TLC1, is deleted, telomeres shorten and most cells die. However, gene conversion mediated by the RAD52 pathway allows telomere lengthening in rare survivor cells. To further investigate the role of recombination in telomere maintenance, we assayed telomere length and the ability to generate survivors in several isogenic DNA recombination mutants, including rad50, rad51, rad52, rad54, rad57, xrs2, and mre11. The rad51, rad52, rad54, and rad57 mutations increased the rate of cell death in the absence of TLC1. In contrast, although the rad50, xrs2, and mre11 strains initially had short telomeres, double mutants with tlc1 did not affect the rate of cell death, and survivors were generated at later times than tlc1 alone. While none of the double mutants of recombination genes and tlc1 (except rad52 tlc1) blocked the ability to generate survivors, a rad50 rad51 tlc1 triple mutant did not allow the generation of survivors. Thus RAD50 and RAD51 define two separate pathways that collaborate to allow cells to survive in the absence of telomerase.     

Differential suppression of DNA repair deficiencies of Yeast rad50, mre11 and xrs2 mutants by EXO1 and TLC1 (the RNA component of telomerase).

Rad50, Mre11, and Xrs2 form a nuclease complex that functions in both nonhomologous end-joining (NHEJ) and recombinational repair of DNA double-strand breaks (DSBs). A search for highly expressed cDNAs that suppress the DNA repair deficiency of rad50 mutants yielded multiple isolates of two genes: EXO1 and TLC1. Overexpression of EXO1 or TLC1 increased the resistance of rad50, mre11, and xrs2 mutants to ionizing radiation and MMS, but did not increase resistance in strains defective in recombination (rad51, rad52, rad54, rad59) or NHEJ only (yku70, sir4). Increased Exo1 or TLC1 RNA did not alter checkpoint responses or restore NHEJ proficiency, but DNA repair defects of yku70 and rad27 (fen) mutants were differentially suppressed by the two genes. Overexpression of Exo1, but not mutant proteins containing substitutions in the conserved nuclease domain, increased recombination and suppressed HO and EcoRI endonuclease-induced killing of rad50 strains. exo1 rad50 mutants lacking both nuclease activities exhibited a high proportion of enlarged, G2-arrested cells and displayed a synergistic decrease in DSB-induced plasmid:chromosome recombination. These results support a model in which the nuclease activity of the Rad50/Mre11/Xrs2 complex is required for recombinational repair, but not NHEJ. We suggest that the 5'-3' exo activity of Exo1 is able to substitute for Rad50/Mre11/Xrs2 in rescission of specific classes of DSB end structures. Gene-specific suppression by TLC1, which encodes the RNA subunit of the yeast telomerase complex, demonstrates that components of telomerase can also impact on DSB repair pathways.     

Inactivation of Escherichia coli by Near-Ultraviolet Light and 8-Methoxypsoralen: Different Responses of Strains B/r and K-12

A series of Escherichia coli K-12 AB1157 strains with normal and defective deoxyribonucleic acid repair capacity were more resistant to treatment with 8-methoxypsoralen (8-MOP) and near-ultraviolet light (NUV) than a comparable series of strains from the B/r WP2 family although sensitivities to 254-nm ultraviolet light were closely similar. The difference was most marked with strains deficient in both excision and postreplication repair (uvrA recA). The hypothesis that the internal level of 8-MOP was lower in K-12 than B/r uvrA recA derivatives was ruled out on the basis of fluorometric determinations of 8-MOP content and the similar inactivation curves for phage T3 treated intracellularly within the two strains. The demonstration of liquid holding recovery with AB2480 but not WP100 (both recA uvrA strains) and the somewhat greater resistance of the former strain to inactivation by captan revealed the presence in the K-12 strain of a deoxyribonucleic acid repair system independent of the recA(+) and uvrA(+) genes. The presence of this repair system did not, however, affect the survival of T3 phage treated with 8-MOP plus NUV and probably has a relatively small effect on survival of AB2480 under normal conditions. Experiments in which 8-MOP monoadducts were converted to cross-links by a second NUV exposure in the absence of 8-MOP indicated that the level of potentially cross-linkable monoadducts immediately after 8-MOP + NUV is about eightfold lower in K-12-than in B/r-derived strains. It is therefore suggested that the photoproduct yield in the former is well below that in the latter. In agreement with this is the observation that, during the first 10 min after treatment, deoxyribonucleic acid synthesis was just over five times more sensitive to inhibition by 8-MOP plus NUV in WP100 than in AB2480. We assume that 8-MOP in K-12 bacteria is hindered in some way from adsorbing to cellular (though not to phage T3) deoxyribonucleic acid. Consistent with this, 8-MOP has been shown to act as an inhibitor of a component of repair of 254-nm ultraviolet light damage in WP2 but not in AB1157.     

Mutagenic effects photoinduced in normal human lymphoblasts by a monofunctional pyridopsoralen in comparison to 8-methoxypsoralen

The photobiological effects induced by the monofuctional 7-methylpyrido[3,4-c]psoralen (MePyPs) in comparison to the bifunctional furocoumarin 8-methoxypsoralen (8-MOP) have been studied in a human lymphoblast cell line TK6. We report that, in human lymphoblasts, the cytotoxic effect of MePyPs plus UVA (365 nm) is much higher than that of 8-MOP plus 365-nm irradiation. The dose-modifying factor at the 37% survival level between the 2 compounds equals 120. Mutation induction by photoactivated MePyPs and 8-MOP has been studied in 2 genetic loci, hypoxanthine phosphoribosyl transferase (HPRT) and Na+/K+ ATPase. For equal UVA doses, the mutagenic effectiveness of MePyPs was higher than that of 8-MOP. However at equal survival levels, the mononfuctional psoralen MePyPs was less efficient than the bifunctional 8-MOP. In other words, compared to 8-MOP, the monofunctional agent MePyPs is more cytotoxic than mutagenic. This higher phototoxic and mutagenic efficiency of MePyPs in comparison to 8-MOP is likely to be related to the chemical nature of MePyPs-induced lesions which may be responsible for a reduced recognition and/or accessibility of the repair enzymes to damaged DNA.     

Repair of endonuclease-induced double-strand breaks in Saccharomyces cerevisiae: essential role for genes associated with nonhomologous end-joining.

Repair of double-strand breaks (DSBs) in chromosomal DNA by nonhomologous end-joining (NHEJ) is not well characterized in the yeast Saccharomyces cerevisiae. Here we demonstrate that several genes associated with NHEJ perform essential functions in the repair of endonuclease-induced DSBs in vivo. Galactose-induced expression of EcoRI endonuclease in rad50, mre11, or xrs2 mutants, which are deficient in plasmid DSB end-joining and some forms of recombination, resulted in G2 arrest and rapid cell killing. Endonuclease synthesis also produced moderate cell killing in sir4 strains. In contrast, EcoRI caused prolonged cell-cycle arrest of recombination-defective rad51, rad52, rad54, rad55, and rad57 mutants, but cells remained viable. Cell-cycle progression was inhibited in excision repair-defective rad1 mutants, but not in rad2 cells, indicating a role for Rad1 processing of the DSB ends. Phenotypic responses of additional mutants, including exo1, srs2, rad5, and rdh54 strains, suggest roles in recombinational repair, but not in NHEJ. Interestingly, the rapid cell killing in haploid rad50 and mre11 strains was largely eliminated in diploids, suggesting that the cohesive-ended DSBs could be efficiently repaired by homologous recombination throughout the cell cycle in the diploid mutants. These results demonstrate essential but separable roles for NHEJ pathway genes in the repair of chromosomal DSBs that are structurally similar to those occurring during cellular development.     

Genotoxic effects and DNA photoadducts induced in Chinese hamster V79 cells by 5-methoxypsoralen and 8-methoxypsoralen

The induction of lethal effects and 6-thioguanine-resistant (6-TGr) mutants were studied in Chinese hamster V79 cells after treatment with the two bifunctional furocoumarins 5- and 8-methoxypsoralens (5-MOP, 8-MOP) in the presence of 365-nm radiation (UVA). The in vivo DNA-photobinding capacity of these two compounds was measured and in parallel the cross-linking capacities of 5-MOP and 8-MOP were determined using the alkaline elution technique. The results show that 5-MOP plus UVA was about 2.5 times more effective than 8-MOP plus UVA for inhibiting cell survival and for inducing the same frequency of 6-TGr mutants (10(-4]. The total number of photoinduced lesions by 5-MOP plus UVA was about 6 times higher than that induced by 8-MOP plus UVA. However, the cross-linking capacities of 5-MOP and 8-MOP were found to be within the same range at equal doses of UVA. At equal number of DNA photoadducts produced, the lesions induced by 5-MOP appeared to be less genetically active than those induced by 8-MOP. The apparently weaker genotoxicity of 5-MOP-induced lesions is likely to be due to the induction of a lower proportion of cross-links by 5-MOP at a given number of photoadducts.