Isolation and genetic characterization of the Neurospora crassa REV1 and REV7 homologs: evidence for involvement in damage-induced mutagenesis

In a previous paper, we reported that the Neurospora crassa upr-1 gene is a homolog of the yeast gene REV3, which encodes the catalytic subunit of DNA polymerase zeta (polzeta). Characterization of the upr-1 mutant indicated that the UPR1 protein plays a role in DNA repair and mutagenesis. To help understand the mechanisms of mutagenic DNA repair in the N. crassa more extensively, we identified N. crassa homologs of yeast REV1 and REV7 and obtained mutants ncrev1 or ncrev7, which had similar phenotypes to the upr-1 mutant. Mutant carrying ncrev7 was more sensitive to UV and 4NQO, and slightly sensitive to MMS than the wild-type. The sensitivity to UV and MMS of the ncrev1 mutant was moderately higher than that of the wild-type, but the sensitivity to 4NQO of the mutant was similar to that of the wild-type. In reversion assay using testers with base substitution or frameshift mutation at the ad-3A locus, each of ncrev1 and ncrev7 mutants showed lower induced-mutability than the wild-type. Expression of ncrev1 and ncrev7 was found to be UV-inducible like the case of upr-1. Genetic analyses showed that the ncrev7 was identical to mus-26, which belongs to the upr-1 epistasis group, and that the ncrev1 was a newly identified DNA repair gene and designated as mus-42. Interestingly, all three mutants have a normal CPD photolyase gene, however, they showed a partial photoreactivation defect (PPD) phenotype, not completely defective but inefficient in photoreactivation. These results suggest that N. crassa REV homolog genes function in DNA repair and UV mutagenesis through the bypass of (6-4) photoproducts.     

Mutagenesis at the ad-3A and ad-3B loci haploid UV-sensitive strains of Neurospora crassa. III. Comparison of dose-response curves for inactivation and mutation induced by gamma-rays

Gamma-Ray-induced inactivation and induction of mutations at the ad-3A and ad-3B loci of Neurospora crassa have been compared among 6 different UV-sensitive strains and a standard wild-type strain. The 6 strains show varying degrees of sensitivity to gamma-ray-induced inactivation, with the relative sensitivity at 37% survival being uvs-6 greater than upr-1 greater than uvs-2 greater than uvs-3 greater than wild-type greater than uvs-5 greater than uvs-4. Studies on the induction of ad-3 mutants by gamma-rays show that when the dose-response curve (expressed in terms of ad-3 mutants among the surviving colonies) of the UV-sensitive strains are compared with wild-type, the 2 excision-repair-deficient mutants uvs-2 and upr-1 exhibit enhanced ad-3 mutant frequencies, uvs-3 exhibits reduced ad-3 mutant frequencies whereas both uvs-4 and uvs-5 show lower mutant frequencies than wild-type.     

Mutagenesis at the ad-3A and ad-3B loci in haploid UV-sensitive strains of Neurospora crassa. II. Comparison of dose-response curves for inactivation and mutation induced by UV

UV-induced inactivation and induction of mutations at the ad-3A and ad-3B loci of Neurospora crassa have been compared among 7 different UV-sensitive strains and a standard wild-type strain. The 7 strains show varying degrees of sensitivity to UV-induced inactivation, with the relative sensitivity being: uvs-2 greater than uvs-3 greater than uvs-4 greater than uvs-6 greater than upr-1 greater uvs-5 greater than uvs-1. Studies on the induction of ad-3 mutants by UV show that the 2 excision-repair deficient mutants uvs-2 and upr-1 exhibit enhanced ad-3 mutant frequencies, while uvs-4 and uvs-5 exhibit reduced ad-3 mutant frequencies, and uvs-3 completely eliminates UV mutagenesis. The ad-3 mutation-induction curves obtained with uvs-1 or uvs-6 are not significantly different from that found with the wild-type strain.     

The upr-1 gene encodes a catalytic subunit of the DNA polymerase zeta which is involved in damage-induced mutagenesis in Neurospora crassa

The upr-1 mutant was one of the first mutagen-sensitive mutants to be isolated in Neurospora crassa. However, the function of the upr-1 gene has not yet been elucidated, although some genetic and biochemical data have been accumulated. In order to clone the upr-1 gene, we performed a chromosome walk from the mat locus, the closest genetic marker to upr-1 for which a molecular probe was available, towards the centromere, and a chromosomal contig of about 300-400 kb was constructed. Some of these clones complemented the temperature sensitivity of the un-16 mutation, which is located between mat and upr-1. The un-16 gene was sequenced, and localized in the MIPS Neurospora crassa genome database. We then searched the regions flanking un-16 for homologs of known DNA repair genes, and found a gene homologous to the REV3 gene of budding yeast. The phenotype of the upr-1 mutant is similar to that of the yeast rev3 mutant. An ncrev3 mutant carrying mutations in the N. crassa REV3 homolog was constructed using the RIP (repeat-induced point mutation) process. The spectrum of mutagen sensitivity of the ncrev3 mutant was similar to that of the upr-1 mutant. Complementation tests between the upr-1 and ncrev3 mutations indicated that the upr-1 gene is in fact identical to the ncrev3 gene. To clarify the role of the upr-1 gene in DNA repair, the frequency of MMS and 4NQO-induced mutations was assayed using the ad-8 reversion test. The upr-1 mutant was about 10 times less sensitive to both chemicals than the wild type. The expression level of the upr-1 gene is increased on exposure to UV irradiation in the uvs-2 and mus-8 mutants, which belong to postreplication repair group, as well as in the wild type. All these results suggest that the product of the upr-1 gene functions in damage-induced mutagenesis and DNA translesion synthesis in N. crassa.     

Ultraviolet-Sensitive Mutator Strain of Escherichia coli K-12

An ultraviolet (UV)-sensitive mutator gene, mutU, was identified in Escherichia coli K-12. The mutation mutU4 is very close to uvrD, between metE and ilv, on the E. coli chromosome. It was recessive as a mutator and as a UV-sensitive mutation. The frequency of reversion of trpA46 on an F episome was increased by mutU4 on the chromosome. The mutator gene did not increase mutation frequencies in virulent phages or in lytically grown phage lambda. The mutU4 mutation predominantly induced transitional base changes. Mutator strains were normal for recombination and host-cell reactivation of UV-irradiated phage T1. They were normally resistant to methyl methanesulfonate and were slightly more sensitive to gamma irradiation than Mut(+) strains. UV irradiation induced mutations in a mutU4 strain, and phage lambda was UV-inducible. Double mutants containing mutU4 and recA, B, or C were extremely sensitive to UV irradiation; a mutU4 uvrA6 double mutant was only slightly more sensitive than a uvrA6 strain. The mutU4 uvrA6 and mutU4 recA, B, or C double mutants had mutation rates similar to that of a mutU4 strain. Two UV-sensitive mutators, mut-9 and mut-10, isolated by Liberfarb and Bryson in E. coli B/UV, were found to be co-transducible with ilv in the same general region as mutU4.     

Mutagenesis at the ad-3A and ad-3B loci in haploid UV-sensitive strains of Neurospora crassa. VI. Genetic characterization of ad-3 mutants provides evidence for qualitative differences in the spectrum of genetic alterations between wild-type and nucleotide excision-repair-deficient strains

Genetic characterization of ad-3B mutants induced in wild-type and UV-sensitive strains has revealed qualitative differences between the spectra of genetic alterations at the molecular level. Ad-3B mutants induced in the two nucleotide excision-repair-deficient strains upr-1 and uvs-2 (Worthy and Epler, 1973) had significantly lower frequencies of nonpolarized complementation patterns and higher frequencies of noncomplementing mutants than ad-3B mutants induced in the wild-type strain in samples induced by either UV, gamma-rays, 4NQO or MNNG. In these same samples ad-3B mutants induced in uvs-4, uvs-5 or uvs-6 did not differ significantly from those induced in the wild-type strain. After ICR-170 treatment, ad-3B mutants induced in the UV-sensitive strains did not differ significantly from those induced in wild-type. The comparisons in the present and previous studies demonstrate that the process of mutation-induction in the ad-3 region is under the control of other loci that not only alter mutant recovery quantitatively (de Serres, 1980; Schüpbach and de Serres, 1981; Inoue et al., 1981a, b) but also qualitatively. These data have important implications for comparative chemical mutagenesis, since the spectrum of genetic alterations produced by a given agent can be modified markedly as a result of defects in DNA repair.     

Biochemical basis of radiation-sensitivity in mutants of Neurospora crassa

The available UV-sensitive mutants of Neurospora crassa were examined for their ability to excise and photoreactive cytosine-containing dimers invivo. All strains exhibited in vivo photoreactivation, including upr-1, which was originally thought to be deficient in photoreactivation. Two strains, uvs-2 and upr-1 were shown to be deficient in excision repair; uvs-3 was shown to contain a residual amount of excision capabilit. The remaining strains, uvs-1, uvs-5, and uvs-6, were normal in their ability to excise dimers. Based on these results, tentative analogies were drawn between the Neurospora mutants and the known classes of UV-sensitive mutants in E. coli. Accordingly, the N. crassa mutants were classified as uvs-1, -lon; uvs-2, -uvr; uvs-3, -uvr (rec?); uvs-5, -lon; uvs-6, -rec; and upr-1, -uvr. A comparison was made between the biochemical responses and the available published data on mutation induction in the Neurospora mutants. Althoughsome relationships were seen between repair defects and mutation induction, too little data were available for any definitive conclusions.     

Effects of heat shock on the induction of mutations by chemical mutagens in Neurospora crassa

Preheating of Neurospora conidia increased their susceptibility to mutation induction by chemical mutagens. Optimal conditions of heat shock for enhanced mutagenesis were determined in 2.5 X 10(7) conidia/ml 0.067 M KH2PO4-Na2HPO4 (pH 7.0) buffer to be treatment at 43 degrees C for 60 min. When protein synthesis during heat stock was eliminated by cycloheximide or by use of the temperature-sensitive mutation psi-1, induction of thermotolerance was inhibited while induction of the enhanced state of mutability was not. Therefore, inducible protein synthesis is not involved in this process. To discover whether DNA-repair systems are altered by heat shock and, as a result, whether reversion frequencies increase, DNA-repair mutants (upr-1, uvs-2, uvs-3, uvs-6, mus-7, mus-16) were heated and their reversion frequencies at the ad-8 locus were measured. All the DNA-repair mutants showed higher reversion frequencies with MNNG treatment after heat shock than in non-heated control. It therefore seems that DNA repair is not involved in the enhancement of chemical mutagenesis by heat shock. Heat shock does not increase frequencies of reversion induced by ultraviolet light, and heat shock after treatment with chemical mutagens does not affect reversion frequencies. These results suggest that heat shock may change the structure and function of cellular membranes and thereby increase the influx of mutagens into cells.     

Mutagen sensitivities and mutator effects of MMS-sensitive mutants in Neurospora

7 mus (mutagen-sensitive) mutants of Neurospora crassa, which are more sensitive to the toxic effects of MMS (methyl methanesulfonate) than wild-type, were investigated for cross-sensitivities to other mutagens and inhibitors. These mutants have recently been mapped in 5 new genes, mus-7 to mus-11, and mutant alleles from each gene were checked for their effects on mutation frequencies. It was found that mutants in 3 of these 5 genes showed radiation-induced mutation frequencies similar to wild-type. These included 2 alleles of the gene mus-10, which were cross-sensitive only to UV and were the only mutants that produced some viable ascospores in homozygous crosses. The mutant of the second gene, mus-8, was especially sensitive to UV and mitomycin C and produced slightly reduced frequencies of spontaneous mutation. In contrast, the mutant of the third gene, mus-7, was not UV-sensitive but showed some cross-sensitivity to X-rays; mus-7 was highly sensitive to MMS and also to histidine, which inhibits various repair-defective mutants at concentrations well below those that reduce wild-type growth. None of these mus resemble mutants previously found in Neurospora, nor do they conform clearly to mutant types identified in E. coli or yeast. On the other hand mutants in 2 further genes, mus-11, and especially 2 alleles of mus-9, are very similar to uvs-3 of Neurospora and generally resemble mutants that are considered to be defective in "error-prone" repair. They were UV- as well as X-ray-sensitive, and showed strong spontaneous mutator effects but almost no increase in recessive lethal frequencies in heterokaryons after UV-treatments.     

Genetic analysis of the Neurospora crassa RAD14 homolog mus-43 and the RAD10 homolog mus-44 reveals that they belong to the mus-38 pathway of two nucleotide excision repair systems

Saccharomyces cerevisiae Rad14 and Rad10 proteins are essential for nucleotide excision repair (NER). Rad14 is a UV-damaged DNA binding protein and Rad10 is a structure-specific endonuclease that functions in a complex with Rad1. In this study, we identified and characterized the RAD14 and RAD10 homolog genes in Neurospora crassa, which we named mus-43 and mus-44, respectively. Disruption of mus-43 and mus-44 conferred sensitivity to UV and 4-nitroquinoline 1-oxide, but not to methyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine, camptothecin, hydroxyurea, or bleomycin. The mus-44 mutant was more sensitive to UV than the mus-43 mutant. Genetic analysis indicated that mus-43 and mus-44 are epistatic to mus-38 which is a homolog of the S. cerevisiae RAD1, but not to mus-18 which belongs to a second excision repair pathway. Immunological assays demonstrated that both mus-43 and mus-44 retained the ability to excise UV-induced cyclobutane pyrimidine dimers and 6-4 photoproducts, but that excision ability was completely abolished in the mus-43 mus-18 and mus-44 mus-18 double mutants. These double mutants exhibited extremely high sensitivity to UV. In mus-43 and mus-44 mutants, the UV-induced mutation frequency increased compared to that of the wild-type. The mus-44 mutants also exhibited a partial photoreactivation defect phenotype similar to mus-38. These results suggest that both mus-43 and mus-44 function in the mus-38 NER pathway, but not in the mus-18 excision repair pathway.     

Mutagenesis at the ad-3A and ad-3B loci in haploid UV-sensitive strains of Neurospora crassa. IV. Comparison of dose-response curves for MNNG, 4NQO and ICR-170 induced inactivation and mutation-induction

The genetic effects of MNNG, 4NQO and ICR-170 have been compared on 5 different UV-sensitive strains and a standard wild-type strain of Neurospora crassa with regard to inactivation and the induction of forward-mutations at the ad-3A and ad-3B loci. Whereas all UV-sensitive strains (upr-1, uvs-2, uvs-3, uvs-5 and uvs-6) are more sensitive to inactivation by MNNG and ICR-170 than wild-type, only uvs-5 shows survival comparable to wild-type after 4NQO treatment, all other strains are more sensitive to 4NQO. In contrast to the effects on inactivation, a wide variety of effects were found for the induction of ad-3A and ad-3B mutations: higher forward-mutation frequencies than were found in wild-type were obtained after treatment with MNNG or 4NQO for upr-1 and uvs-2, no significant increase over the spontaneous mutation frequency was found with uvs-3 after MNNG, 4NQO or ICR-170 treatment; mutation frequencies comparable to that found in wild-type were obtained with uvs-6 after MNNG, 4NQO or ICR-170 treatment and with upr-1 after ICR-170 treatment. Lower forward-mutation frequencies than were found in wild-type were obtained with uvs-2 after ICR-170 treatment and with uvs-5 after MNNG, 4NQO or ICR-170 treatment. These data clearly show that the process of forward-mutation at the ad-3A and ad-3B loci is under genetic control by mutations at other loci (e.g. upr-1, uvs-2, uvs-3, uvs-5 and uvs-6) and that the effect is markedly mutagen-dependent.     

A novel phenotype of an excision-repair mutant in Neurospora crassa: mutagen sensitivity of the mus-18 mutant is specific to UV.

Summary A UV-sensitive mutant has been isolated from UV-mutagenized conidia of Neurospora crassa. The mutation responsible for the lesion was mapped in linkage group VL, proximal to the nucleolus organizer region. We designated the mutant mus-18. The sensitivity of the mus-18 mutant to UV-irradiation was not particularly high, being less than twice that of the wild-type strain. However, the frequency of mutations at the ad-3 loci induced by UV was extremely high even at low doses, under conditions where survival rates of mus-18 cells were almost identical to those of wild-type cells. Photoreactivation of UV damage was normal in the mus-18 mutant. Sensitivity to other mutagens, such as gamma rays, 4-nitroquinoline-1-oxide, N-methyl-N-nitro-N-nitrosoguanidine, mitomycin C and methyl methanesulfonate, was similar to that of the wild type. Fertility of the mus-18 mutant was normal in homozygous crosses. These results suggest that mus-18 is an excision-repair mutant. Measurement of endonuclease-sensitive sites (ESS) after liquid-holding recovery from UV damage revealed that ESS remained unrepaired for longer than 18 h in the mus-18 mutant, while most were eliminated within 6 h in wild-type cells and in other UV-sensitive mutants. This result suggests that mus-18 is defective in the incision step of dimer excision. The mus-18 mutant provides the first example of an excision-defective mutation in eukaryotes, which is specific to UV damage.     

Inactivation of carotenoid-producing and albino strains of Neurospora crassa by visible light, blacklight, and ultraviolet radiation.

Suspensions of Neurospora crassa conidia were inactivated by blacklight (BL) radiation (300 to 425 nm) in the absence of exogenous photosensitizing compounds. Carotenoid-containing wild-type conidia were less sensitive to BL radiation than albino conidia, showing a dose enhancement factor (DEF) of 1.2 for dose levels resulting in less than 10% survival. The same strains were about equally sensitive to shortwave ultraviolet (UV) inactivation. The kinetics of BL inactivation are similar to those of photodynamic inactivation by visible light in the presence of a photosensitizing dye (methylene blue). Only limited inactivation by visible light in the absence of exogenous photosensitizers was observed. BL and UV inactivations are probably caused by different mechanisms since wild-type conidia are only slightly more resistant to BL radiation (DEF = 1.2 at 1.0% survival) than are conidia from a UV-sensitive strain (upr-1, uvs-3). The BL-induced lethal lesions are probably no cyclobutyl pyrimidine dimers since BL-inactivated Haemophilus influenzae transforming deoxyribonucleic acid is not photoreactivated by N. crassa wild-type enzyme extracts, whereas UV-inactivated transforming deoxyribonucleic acid is photoreactivable with this treatment.     

A eukaryotic gene encoding an endonuclease that specifically repairs DNA damaged by ultraviolet light.

Many eukaryotic organisms, including humans, remove ultraviolet (UV) damage from their genomes by the nucleotide excision repair pathway, which requires more than 10 separate protein factors. However, no nucleotide excision repair pathway has been found in the filamentous fungus Neurospora crassa. We have isolated a new eukaryotic DNA repair gene from N.crassa by its ability to complement UV-sensitive Escherichia coli cells. The gene is altered in a N.crassa mus-18 mutant and responsible for the exclusive sensitivity to UV of the mutant. Introduction of the wild-type mus-18 gene complements not only the mus-18 DNA repair defect of N.crassa, but also confers UV-resistance on various DNA repair-deficient mutants of Saccharomyces cerevisiae and a human xeroderma pigmentosum cell line. The cDNA encodes a protein of 74 kDa with no sequence similarity to other known repair enzymes. Recombinant mus-18 protein was purified from E.coli and found to be an endonuclease for UV-irradiated DNA. Both cyclobutane pyrimidine dimers and (6-4)photoproducts are cleaved at the sites immediately 5' to the damaged dipyrimidines in a magnesium-dependent, ATP-independent reaction. This mechanism, requiring a single polypeptide designated UV-induced dimer endonuclease for incision, is a substitute for the role of nucleotide excision repair of UV damage in N.crassa.     

DNA Repair Dependence of Somatic Mutagenesis of Transposon-Caused WHITE Alleles in DROSOPHILA MELANOGASTER after Treatment with Alkylating Agents

DNA repair-defective alleles of the mei-9, mei-41, mus-104 and mus-101 loci of Drosophila melanogaster were introduced into stocks bearing the UZ and SZ marker sets. Males with the UZ marker set, z1 (zeste allele) and w+(TE) (genetically unstable white allele presumably caused by a transposable element), or the SZ marker set, z1 and w+R (semistable white allele caused by partial duplication of the w+ locus plus transposon insert), were exposed to EMS at the first instar. After emergence, adult males bearing red spots on lemon-yellow eyes were scored as flies with somatic reversions of w+(TE) or w+R. The relative mutabilities (relative values of reversion frequency at an equal EMS dose) of either w+(TE) or w+R in a repair-proficient strain and in mei-9, mei-41, mus-104 and mus-101 strains were 1: approximately 1.2:0.3:0.3:0.7, despite the fact that w+(TE) reverted two to three times as frequently as w+R under both the repair-proficient and repair-deficient genetic conditions. Similarly, after treatment with MMS, MNNG and ENNG, w+(TE) was somatically more mutable in the mei-9 strain and less mutable in the mei-41 and mus-104 strains than in the repair-proficient strain. From these results, we propose that mutagenic lesions produced in DNA by treatment with these chemicals are converted to mutant DNA sequences via the error-prone repair mechanisms dependent on the products of the genes mei-41+ (mei-41 and mus-104 being alleles of the same locus) and mus-101+, whereas they are eliminated by mei-9+-dependent excision repair. In contrast to the approximately linear responses of induced reversions of w+(TE) with ENNG in the repair-proficient, mei-9, and mei-41 strains, seemingly there were dosage insensitive ranges for induced reversion with MNNG in the repair-proficient and mei-41 strains, but not for reversion in the mei-9 strain; w+(TE) in the mus-104 strain was virtually nonmutable with MNNG and ENNG. These results suggest that O6-methylguanine (O6MeG) produced in DNA with MNNG, but not O6-ethylguanine produced with ENNG, is almost completely repaired in a low dose range by constitutive activity of DNA O6MeG transmethylase. From the distribution of clone sizes of spontaneous revertant spots and other data, we propose that both w+(TE) and w+R have a similar tendency to spontaneously revert more frequently at early rather than at later developmental stages probably reflecting a common property of their inserted transposons.     

A UV-supersensitive mutant in the yeast Schizosaccharomyces pombe

Summary A high UV-sensitive mutant was obtained from a UV-sensitive strain of the yeast Schizosaccharomyces pombe after a mutagenic treatment. By genetic analysis, it was possible to distinguish two independent loci. The double mutant is supersensitive, that is more UV-sensitive than either of the two single mutants. This suggests that the mutations involved interfere with two repair pathways that are, at least partially, independent of each other.Some properties of the two single mutants were studied. These mutants differ notably in their response to caffeine, to liquid-holding, to exposure to visible light after UV irradiation, and in their UV-sensitive during the logarithmic growth phase.Comparison of the properties of the wild-type strain and of the different UV sensitive mutants leads to the conclusion that one repair pathway is used preferentially in the wild-type strain.Abbreviations DRF dose reduction factor - LH liquid holding     

Effect of degradative plasmid CAM-OCT on responses of Pseudomonas bacteria to UV light.

The effect of plasmid CAM-OCT on responses to UV irradiation was compared in Pseudomonas aeruginosa, in Pseudomonas putida, and in Pseudomonas putida mutants carrying mutations in UV response genes. CAM-OCT substantially increased both survival and mutagenesis in the two species. P. aeruginosa strains without CAM-OCT exhibited much higher UV sensitivity than did P. putida strains. UV-induced mutagenesis of plasmid-free P. putida was easily detected in three different assays (two reversion assays and one forward mutation assay), whereas UV mutagenesis of P. aeruginosa without CAM-OCT was seen only in the forward mutation assay. These results suggest major differences in DNA repair between the two species and highlight the presence of error-prone repair functions on CAM-OCT. A number of P. putida mutants carrying chromosomal mutations affecting either survival or mutagenesis after UV irradiation were isolated, and the effect of CAM-OCT on these mutants was determined. All mutations producing a UV-sensitive phenotype in P. putida were fully suppressed by the plasmid, whereas the plasmid had a more variable effect on mutagenesis mutations, suppressing some and producing no suppression of others. On the basis of the results reported here and results obtained by others with plasmids carrying UV response genes, it appears that CAM-OCT may differ either in regulation or in the number and functions of UV response genes encoded.     

Genetic analysis of CHK1 and CHK2 homologues revealed a unique cross talk between ATM and ATR pathways in Neurospora crassa

DNA damage checkpoint is an important mechanism for organisms to maintain genome integrity. In Neurospora crassa, mus-9 and mus-21 are homologues of ATR and ATM, respectively, which are pivotal factors of DNA damage checkpoint in mammals. A N. crassa clock gene prd-4 has been identified as a CHK2 homologue, but its role in DNA damage response had not been elucidated. In this study, we identified another CHK2 homologue and one CHK1 homologue from the N. crassa genome database. As disruption of these genes affected mutagen tolerance, we named them mus-59 and mus-58, respectively. The mus-58 mutant was sensitive to hydroxyurea (HU), but the mus-59 and prd-4 mutants showed the same HU sensitivity as that of the wild-type strain. This indicates the possibility that MUS-58 is involved in replication checkpoint and stabilization of stalled forks like mammalian CHK1. Phosphorylation of MUS-58 and MUS-59 was observed in the wild-type strain in response to mutagen treatments. Genetic relationships between those three genes and mus-9 or mus-21 indicated that the mus-9 mutation was epistatic to mus-58, and mus-21 was epistatic to prd-4. These relationships correspond to two signal pathways, ATR-CHK1 and ATM-CHK2 that have been established in mammalian cells. However, both the mus-9 mus-59 and mus-21 mus-58 double mutants showed an intermediate level between the two parental strains for CPT sensitivity. Furthermore, these double mutants showed severe growth defects. Our findings suggest that the DNA damage checkpoint of N. crassa is controlled by unique mechanisms.     

Isolation and Characterization of Ultraviolet Light-Sensitive Mutants of the Blue-Green Alga Anacystis nidulans

Three independently isolated ultraviolet light-sensitive (uvs) mutants of Anacystis nidulans were characterized. Strain uvs-1 was most sensitive to UV in the absence of photoreactivation. Pretreatment with caffeine suppressed the dark-survival curve of strain uvs-1, indicating the presence of excision enzymes involved in dark repair. Under "black" and "white" illumination, strain uvs-1 displays photoreactivation properties nearly comparable to wild-type culture. Mutants uvs-35 and uvs-88 appeared to have partial photorecovery capacities. Upon pretreatment with chloramphenicol, photoreactivation properties of strains uvs-1 and uvs-88 were not evident although the partial photoreactivation characteristics of strain uvs-35 remained the same. Data indicate that strains uvs-1, uvs-35, and uvs-88 are probably genetically distinct UV-sensitive mutants.     

Isolation of recombination-defective and UV-sensitive mutants of Bacillus megaterium.

Mutants of Bacillus megaterium QMB1551 sensitive to mitomycin C or methyl methanesulfonate were isolated and characterized phenotypically. Cell survival after UV-light and gamma-ray exposure was determined, as was transductional recombination. Of the mutants tested, three were sensitive to UV but remained recombination proficient. The UV-sensitive mutants were also reduced in host cell reactivation. At least three mutants had undetectable transduction frequencies, i.e., less than 0.3 to 1.3% of the parental strain frequencies, and so appear to be recombination deficient. Sensitivities of these mutant strains to UV light and gamma radiation were compared with those of parental B. megaterium as well as parental, recE4, recA1, uvrA19, and uvrB109 strains of Bacillus subtilis. In each case, the strains of B. megaterium, including the parental strains, showed a higher percentage of cell survival than B. subtilis.