Non-essential UV-sensitive bacteriophage T4 mutants affecting early DNA synthesis: a third pathway of DNA repair.

Non-essential bacteriophage T4 mutants uvs58 and uvs79 showed a lower UV sensitivity than either the excision-repair mutant v am5 or the replication-dependent recombination-repair mutant y10. The UV sensitivity of double and triple mutants carrying one of the mutations uvs58 or uvs79, and v am 5 or (and) y10 was higher than the sum of the sensitivities of the single mutants. The uvs58 mutation was mapped to the early gene region, close to amN81 (gene 41). The unirradiated mutants uvs58 and uvs79 accumulated newly synthesized DNA at a slower rate than wild-type T4. Double mutants uvs58:am59 and uvs79:am59 showed DNA synthesis in E. coli B su- to be arrested at a 3--5 times lower level than that in am59-infected cells. Chloramphenicol, added 9--12 min after infection, suppressed arrests of DNA synthesis, the double mutants showing a lag of 8 min as compared with am59. Results from analysis of sucrose gradients of parental uvs58 and uvs79 DNA were in agreement with the suggestion of a mutation in an early function. The mutants uvs58 and uvs79 are suggested to be defective in a component of the DNA replication apparatus with a function in the adaptation to irregularities in the DNA structure. The third pathway of UV repair is tentatively designated as non-catalytic replication repair.     

Genetic control of the sensitivity of Aspergillus nidulans to mutagenic factors. VII. Inheritance of cross-sensitivity to different mutagenic factors by uvs-mutants]

To study the inheritance of the sensitivity to UV, X-rays, methylmethanesulphonate (MMS), nitrosoguanidine (NG) and nitrous acid (NA) in five uvs mutants of Aspergillus nidulans, having multiple sensitivity to these factors, the sensitivity of recombinants obtained from crossing uvs mutants with uvs+ strain, resistant to all the factors analysed, and uvs leads to uvs+ revertants is investigated. Four uvs mutants (15, 17, 19 and 26) are found to have a nomogenic control of sensitivity to different mutagens. In one mutant (uvs11) the sensitivity to five factors is controlled by two non-linked mutations, one of them determining the sensitivity to UV, NG, NA, and the other--to X-rays and MMC. Phenotypic manifestations of uvs mutations is modified by cell genotype, both chromosomal and cytoplasmic factors being responsible for the modification. Phenotypic modification of uvs mutation results in the change to some (but not to all) mutagenic factors. It suggests, that not the product of uvs gene, but some other components of the reparation complex are modified. Otherwise, reparation of different DNA damages can be carried out by a single enzyme acting in different reparation complexes.     

UV-induced DNA degradation in Aspergillus nidulans cells]

UV-induced DNA degradation was studied in mycellial cells of Aspergillus nidulans wild type and several uvs mutants. It was shown to be an enzymatic specific process which possibly reflects the excision of pyrimidine dimers from UV-damaged DNA. Inhibition of DNA degradation by caffeine and 2,4-dinitrophenol shows the connection between degradation and repair of DNA. Two ways of DNA degradation were found in A. nidulans cells, one of them being glucose dependent and the other--glucose independent. The dependence of DNA degradation on protein synthesis before and after UV-irradiation was demonstrated. The scheme of ways of DNA degradation and its genetic control were suggested on the basis of uvs mutations effect on UV-induced DNA degradation.     

A New Epistasis Group for the Repair of DNA Damage in Bacteriophage T4: Replication Repair

The gene 32 mutation amA453 sensitizes bacteriophage T4 to the lethal effects of ultraviolet (UV) irradiation, methyl methanesulfonate and angelicin-mediated photodynamic irradiation when treated particles are plated on amber-suppressing host cells. The increased UV sensitivity caused by amA453 is additive to that caused by mutations in both the T4 excision repair (denV) and recombination repair (uvsWXY) systems, suggesting the operation of a third kind of repair system. The mutation uvs79, with many similarities to amA453 but mapping in gene 41, is largely epistatic to amA453. The mutation mms1, also with many similarities to amA453, maps close to amA453 within gene 32 and is largely epistatic to uvs79. Neither amA453 nor uvs79 affect the ratio of UV-induced mutational to lethal hits, nor does amA453 affect spontaneous or UV-enhanced recombination frequencies. Gene 32 encodes the major T4 ssDNA-binding protein (the scaffolding of DNA replication) and gene 41 encodes a DNA helicase, both being required for T4 DNA replication. We conclude that a third repair process operates in phage T4 and suggest that it acts during rather than before or after DNA replication.     

Genetic control of recombination processes in Aspergillus nidulans. I. Effect of uvs-mutations on the frequency of spontaneous and ultraviolet ray-induced intergenic recombination]

Effect of 3 uvs mutations (uvs 12, 19 and 25) on recombination processes in Aspergillus nidulans is studied. All the mutations are found either to affect the fertility of carp bodies and germination ability of askospores, or result in complete inability of heterokaryons to form cleistocarpia. Two mutations change the frequency of spontaneous meitotic crossing-over at pro-paba region of the chromosome I and do not affect the rate of mitotic recombination at w-centromeric region of the chromosome II: uvs 12 mutation increases, and uvs 19 mutation decreases the frequency of meiotic recombination. One mutation (uvs 25) decreases the rate of spontaneous mitotic crossing-over. All uvs mutations decrease the frequency of VU light induced mitotic recombination at w-centromeric region of the chromosome II. The data obtained, together with earlier reported characteristics of uvs mutants, suggest that recombination mechanisms in yeast participate in reparation processes more actively than in prokariotes. Different effects of the same uvs mutations on spontaneous frequency of meiotic and mitotic crossing-over draw to the conclusion that genetic control and molecular mechanisms of these processes in A. nidulans are not identical.     

Genetic analysis of DNA repair in Aspergillus: evidence for different types of MMS-sensitive hyperrec mutants

To identify genes which affect DNA repair and possibly recombination in Aspergillus nidulans, mutants hypersensitive to methyl methanesulphonate (MMS) were induced with ultraviolet light (UV) or gamma-rays. About half of them contained associated translocations and many were hypersensitive to UV and/or defective in meiosis. Two are alleles of the known uvsB gene while most others define new genes. In addition, among available uvs mutants many were found to be MMS-sensitive. Some of the various uncharacterized ones were identified as alleles of known uvs, but 5 of them were mapped in 2 new genes, uvsH and uvsJ. To identify functional and epistatic groups, mutants from each uvs gene were tested for effects on recombination and mutation, and double mutant uvs strains were compared for UV survival to their component single mutant strains. 3 epistatic pairs were identified, (1) uvsF and H, (2) uvsB and D, and (3) uvsC and E. Conclusive interpair tests were difficult, because such double mutant combinations were frequently lethal or nearly so. The first pair, uvsF and H, shared some of the properties of excision-defective mutants, both uvs being very highly sensitive to UV for mutation as well as survival. But unlike such mutants, uvsH was also sensitive to gamma-rays and defective in meiosis. Both uvs showed normal levels of meiotic recombination, but greatly increased spontaneous mitotic crossing-over, being the most "hyperrec" types among all uvs. The second pair, uvsB and uvsC, which was similarly hyperrec showed only slight increases of UV-induced mutation (less than 2-fold). As a main effect, these uvs caused very high frequencies of unbalanced, unstable segregants from diploid conidia (30 X), but few of these were recognizable aneuploids. The third pair, uvsC and E, which are known to be rec- for gene conversion, caused reduced mitotic crossing-over in diploids and increased levels of haploid segregants. These mutants are spontaneous mutators, but showed less UV-induced mutation than wild-type controls.     

RAD29 and RAD31--new genes from Saccharomyces cerevisiae yeasts, participating in control of DNA repair. Isolation and genetic study of mutants

Base excision repair (BER) and nucleotide excision repair (NER) are two main cellular responses to DNA damage induced by various physical and chemical factors. After exposure of the strain that carries the NER-blocking rad2 mutation to UV light, several mutants hypersensitive to the UV light lethal action and simultaneously sensitive to methylmethanesulphonate (MMS) were isolated. Two of these mutants (Uvs64 and Uvs212) were examined in detail. The mutants were found to carry recessive, monogenically inherited lesions that had pleiotropic, though different, phenotypes: both mutants were also sensitive to nitrous acid (HNO2), whereas Uvs212 was sensitive to hydrogen peroxide as well. Moreover, the homozygote for the uvs212 mutation, but not for uvs64, blocks the sporulation. Since the mutations examined were not allelic to any of the known rad mutations that cause MMS sensitivity or to each other, it is concluded that two new genes involved in the control of yeast DNA repair were detected. Furthermore, these genes were mapped to different regions of the right arm of chromosome 2 where repair genes were not found. Thus, two new genes, designated RAD29(UVS64) and RAD31(UVS212) and probably involved in base excision repair, were identified.     

Electiveness of photorepair, influence of dark-repair on shape of dose-response curves, and high-dose decline, in UV-induced colour mutations of Serratia.

Strain CV of Serratia marcescens mutates by UV with high frequency to 3 groups of mutants (w, h, s) differing in colour from the red wild-type. The mutational dose—response curve has a curvature corresponding to about 3 hits. It reaches a peak and declines at high doses. Inactivation curves have a broad shoulder and mostly, but not always, a break to a lesser slope at UV doses near the peak of mutations. Photoreactivation (PR) gives a dose reduction of about 2 for both inactivation and mutation including the break and peak. The dose curve with PR for w-mutations shows 1 hit-, and the other types 2-hit curvature leading to a change of mutation spectrum with dose due to PR. The UV-sensitive mutant uvs21 of CV has a survival curve with a small shoulder and a long upward concavity without a break, and the mutation curve is of the one-hit type without a peak and decline. PR gives a dose reduction of 12 for inactivation and of 7.5 for mutation. The 3-hit mutation curve of CV is interpreted by assuming that 2 further hits are required to protect the 1-hit pre-mutations from being abolished by the repair lacking in uvs21. UV induction of SOS repair cannot be responsible for the 3-hit curvature because UVR of phages and induction of prophage are already saturated at rather low doses. High-dose decline (HDD) of mutations in CV is probably caused neiher by a fraction of UV-resistant cells in the population nor by post-mutational selective inhibition of growth of mutants by UV-inactivated neighbour cells. As HDD is not observed in uvs21, possibly the non-mutagenic repair lacking from uvs21 interferes with the mutation finishing processes at high doses in the repair-proficient strain CV. However, UV induction of this interference cannot be a one-hit process but requires a very large number of hits.     

Aberrant DNA repair and enhanced mutagenesis following mutagen treatment of Chinese hamster Ade-C cells in a state of purine deprivation

Ade-C is a Chinese hamster ovary cell line auxotrophic for purines because of a mutation in the de novo synthetic pathway. We now show that, in the absence of exogenous hypoxanthine, replicative DNA synthesis is rapidly shut down. Various aspects of DNA repair have been studied in purine-starved cells. Incision, the first step of excision repair of UV damage, appears normal, as do the later steps, repair synthesis (demonstrated following chemical damage as well as UV-irradiation) and ligation. However, removal of UV-induced pyrimidine dimers is not detected, and it seems that the repair that occurs is aberrant. This behaviour is associated with an increase in cell killing by UV light, and a several-fold increase in the frequency of mutations induced by UV.     

Aberrant DNA repair and enhanced mutagenesis following mutagen treatment of Chinese hamster Ade−C cells in a state of purine deprivation

Ade⁻C is a Chinese hamster ovary cell line auxotrophic for purines because of a mutation in the de novo synthetic pathway. We now show that, in the absence of exogenous hypoxanthine, replicative DNA synthesis is rapidly shut down. Various aspects of DNA repair have been studied in purine-starved cells. Incision, the first step of excision repair of UV damage, appears normal, as do the later steps, repair synthesis (demonstrated following chemical damage as well as UV-irradiation) and ligation. However, removal of UV-induced pyrimidine dimers is not detected, and it seems that the repair that occurs is aberrant. This behaviour is associated with an increase in cell killing by UV light, and a several-fold increase in the frequency of mutations induced by UV.     

UV-induced recessive lethals in uvs strains of Neurospora which are deficient in UV mutagenesis

The frequencies of spontaneous and UV-induced recessive lethal mutations were compared for UV-sensitive and wild-type heterokaryons of Neurospora crassa. These heterokaryons were homokaryotic either for one of two alleles of uvs-3, or for uvs-6 or uvs+. For uvs-3, which is known to have mutator effects, spontaneous recessive lethals were found to be 4-6 times more frequent than observed in uvs+. After correction for clonal distribution of spontaneous mutants, an observed 2-fold increase for uvs-6 was not statistically significant and may have been due to chance occurrence of a few large clones of mutants. Treatment with low doses of UV (50-200 J/m2) produced very similar overall rates of increase for recessive lethals in uvs and uvs+ heterokaryons. This means, that in contrast to results obtained when mutation to ad-3 was measured, both uvs-3 alleles showed highly significant increases for recessive lethals when treated with UV. It is proposed that certain types of UV damage may be processed into recessive lethal mutations by an alternate mechanism from that responsible for viable mutations.     

Induction of gene mutations and the lethal action of ultraviolet rays in synchronized Chinese hamster cells

Lethal and mutagenic effects of UV light were studied in two synchronized UV-sensitive Chinese hamster cell clones differing in the degree of sensitivity (CHS1, CHS2). It is shown that the phase of mitosis is most resistant to the lethal effect of UV. The sensitivity of both cell clones increases in the pre-synthetic phase and reaches its maximum during the phase of DNA synthesis. Positive correlation of cell sensitivity to mutagenic and lethal action of UV was observed when studying induced mutability in both cell clones during the phase of DNA synthesis. However, the study of the mutagenic effect of UV on different phases of the synthesis. However, the study of the mutagenic effect of UV on different phases of the cell cycle (M, G1, S) in the less UV-sensitive cell clone has revealed that the maximal mutation yield takes place when cells are irradiated at G1 (CHS1). The discrepancy observed may be due to different probability of the phenotypic detection of pre-mutational lesions, arising at different phases of the cell cycle. It is shown that only one cell generation is necessary for the expression of pre-mutational changes. These data allow to conclude that the increased mutation rate observed at G1 (as compared with S) reveals rather a probability of the expression but not of the occurrence of pre-mutational lesions. It is suggested that the fixation of mutations in the cells studied proceeds during the post-replication repair synthesis.     

Hypermutation rate normalized by chronological time

It is generally believed that in cells undergoing Ig somatic hypermutation, more cell divisions result in more mutations. This is because DNA synthesis and replication is thought to play roles in the known mechanisms-cytidine deamination and subsequent conversion to thymidine, uracil-DNA glycosylase-mediated repair, mismatch repair, and DNA synthesis by error-prone polymerases. In this study, we manipulated the number of cell generations by varying the rate at which cultures of a mouse cell line were replenished with fresh medium. We found that the frequency of mutants does not necessarily increase with the number of cell generations. On the contrary, a greater number of divisions can lead to a lower frequency of mutants, indicating that cell division is not a rate-limiting step in the hypermutation process. Thus, when comparing mutation rates, we suggest that rates are more appropriately expressed as mutations per day than per cell generation.     

Temporal genetic mapping in the blue-green alga Anacystis nidulans using ethyl methanesulphonate.

Cultures of the blue-green alga Anacytis nidulans were synchronized with respect to DNA synthesis as well as cell division. Application of ethyl methanesulphonate at different stages of replication resulted in a peak of mutation frequency for different genetic markers; this peak can be accounted for in terms of the involvement of repair processes. A temporal map of 19 markers has been constructed by this method. Comparison of gene position obtained by temporal mapping indicates that either bidirectional replication or unidirectional replication from more than one origin occurs.     

Ultraviolet light-induced mutation in UV-resistant, thermosensitive derivatives of lexA-strains of Escherichia coli K-12

It was shown previously that a major class of UV-resistant derivatives of lexA- strains of E. coli K-12 is defective in cell division at 42.5 degrees. The thermosensitive mutations, judging by genetic mapping and complementation tests, are believed to be intragenic suppressor mutations that lower the activity of the diffusible product that results in the LexA- phenotype (Mount et al., 1973). Several thermosensitive derivatives have been characterized in regard to their susceptibility to mutation induction by UV at the permissive growth temperature (30 degrees). Although the strains tested are approximately as resistant to UV as lexA+ strains, they showed a level of mutation induction that was considerably lower. By means of genetic complementation tests it was demonstrated that the low levels of UV mutagenesis in lexA- strains and their thermosensitive derivatives result from the synthesis of a diffusible product. One possible interpretation of these results is that a diffusible product in lexA- strains prevents the induction of error-prone repair. Altering the activity of this product by tsl mutations can lead to increased, but not normal, levels of error-prone repair.     

Effects of multiple yeast rad3 mutant alleles on UV sensitivity, mutability, and mitotic recombination.

A yeast strain was constructed that had a disruption of the chromosomal RAD3 gene and carried a series of centromeric plasmids with defined mutations in this gene. Using this isogenic collection, we examined sensitivity to UV radiation, spontaneous and UV radiation-induced mutagenesis, and mitotic recombination. Several alleles resulted in a marked increase in UV sensitivity. Most of these alleles were found to carry mutations located in consensus motifs for DNA helicases. Other alleles caused a modest or no increase in UV sensitivity and carried mutations in regions of the Rad3 polypeptide that are apparently not conserved. This correlation suggests that the DNA helicase activity of Rad3 protein is required for nucleotide excision repair of DNA. Some rad3 alleles conferred a marked increase in the frequency of spontaneous mutagenesis, including nonsuppressor reversion of the lys2-1 ochre mutation. These alleles also showed a good correlation with conserved DNA helicase domains, suggesting that the Rad3 DNA helicase also plays a role in the fidelity of DNA synthesis or postreplicative mismatch correction. Several rad3 mutator alleles also resulted in increased levels of mitotic recombination. Increased spontaneous mutagenesis and mitotic recombination are characteristic features of the Rem- phenotype. However, in contrast to the prototypic Rem- phenotype, the rad3 mutator alleles identified in this study did not confer inviability in the presence of mutations in the RAD50 or RAD52 gene required for strand break repair of DNA.     

A point mutation in the Aspergillus nidulans sonBNup98 nuclear pore complex gene causes conditional DNA damage sensitivity

The nuclear pore complex (NPC) is embedded in the nuclear envelope where it mediates transport between the cytoplasm and nucleus and helps to organize nuclear architecture. We previously isolated sonB1, a mutation encoding a single amino acid substitution within the Aspergillus nidulans SONBnNup98 NPC protein (nucleoporin). Here we demonstrate that this mutation causes marked DNA damage sensitivity at 42 degrees . Although SONBnNup98 has roles in the G2 transition, we demonstrate that the G2 DNA damage checkpoint is functional in the sonB1 mutant at 42 degrees . The MRN complex is composed of MRE11, RAD50, and NBS1 and functions in checkpoint signaling, DNA repair, and telomere maintenance. At 42 degrees we find that the DNA damage response defect of sonB1 mutants causes synthetic lethality when combined with mutations in scaANBS1, the A. nidulans homolog of NBS1. We provide evidence that this synthetic lethality is independent of MRN cell cycle checkpoint functions or MREAMRE11-mediated DNA repair functions. We also demonstrate that the single A. nidulans histone H2A gene contains the C-terminal SQE motif of histone H2AX isoforms and that this motif is required for the DNA damage response. We propose that the sonB1 nucleoporin mutation causes a defect in a novel part of the DNA damage response.     

The Large Subunit of Replication Factor C (Rfc1p/Cdc44p) Is Required for DNA Replication and DNA Repair in Saccharomyces Cerevisiae

We used genetic and biochemical techniques to characterize the phenotypes associated with mutations affecting the large subunit of replication factor C (Cdc44p or Rfc1p) in Saccharomyces cerevisiae. We demonstrate that Cdc44p is required for both DNA replication and DNA repair in vivo. Cold-sensitive cdc44 mutants experience a delay in traversing S phase at the restrictive temperature following alpha factor arrest; although mutant cells eventually accumulate with a G2/M DNA content, they undergo a cell cycle arrest and initiate neither mitosis nor a new round of DNA synthesis. cdc44 mutants also exhibit an elevated level of spontaneous mutation, and they are sensitive both to the DNA damaging agent methylmethane sulfonate and to exposure to UV radiation. After exposure to UV radiation, cdc44 mutants at the restrictive temperature contain higher levels of single-stranded DNA breaks than do wild-type cells. This observation is consistent with the hypothesis that Cdc44p is involved in repairing gaps in the DNA after the excision of damaged bases. Thus, Cdc44p plays an important role in both DNA replication and DNA repair in vivo.     

Adaptation to cycloheximide of macromolecular synthesis in Tetrahymena

Cycloheximide (CHI) at 10 ng/ml partially inhibited protein synthesis in exponential cultures of Tetrahymena Sp. At 20 ng/ml or greater, inhibition was complete. When protein synthesis was inhibited to any extent, cell division ceased immediately. In all instances where measured, synthesis of RNA and DNA also ceased. After a period of delay, cellular functions reinitiated in the order: (i) protein synthesis, (ii) DNA synthesis and, (iii) RNA synthesis and cell division. The delay in cell division was divided into three phases of: I, zero; II, low; and, III, fully recovered rates of exponential protein synthesis. The length of the three phases increased with increasing concentration of CHI Prior growth of cells for one generation in the presence of 7.5 ng/ml CHI (facilitation) eliminated phase I and slightly decreased phases II and III following subsequent challenge with an inhibitory concentration of CHI. Facilitation for six generations further decreased phases II and III. Protein synthesis and cell division were not inhibited during facilitation In the culture, succinate dehydrogenase activity did not increase during the delay but increased normally at the onset of division. In contrast, NADPH-cytochrome c reductase activity continued to increase for an hour after inhibition of protein synthesis, was constant for a period and did not increase again until an hour after reinitiatoin of cell division and RNA synthesis Inhibition of division of all cells was immediate and reinitiation of synthesis and cell division was non-synchronous.     

Pollen DNA repair after treatment with the mutagens 4-nitroquinoline-1-oxide, ultraviolet and near-ultraviolet irradiation, and boron dependence of rapair

Summary Irradiation of dry, mature pollen from Petunia hybrida with near-ultraviolet light from an erythemal-sunlamp gave rise to a repair-like, unscheduled DNA synthesis during the early stages of in vitro germination. Like that brought about by farultraviolet light from a germicidal lamp, this DNA synthesis is enhanced by hydroxyurea added to the germination medium, and reduced by photoreactivating light given after ultraviolet irradiation and before germination begins. It is concluded that pollen, often receiving considerable exposure to sunlight, has, in addition to the protection afforded by the ultraviolet filtering effect of yellow pigments, also the capacity to repair ultraviolet produced changes in DNA, by both photoreactivation and dark repair processes.Because mature Petunia pollen is arrested at the G₂ stage of the cell cycle, germinating pollen provides us with a highly synchronous plant tissue with a very low background of DNA replicative synthesis suitable for sensitive measurement of DNA repair synthesis. Thus we have shown that 4-nitroquinoline-1-oxide, at concentrations greater than 0.001 mM, gives rise to an unscheduled DNA synthesis which is enhanced by hydroxyurea. Like that induced by ultraviolet radiation, the chemical mutagen brings about DNA repair only during the early stages of pollen germination, and further it has been possible to show that repair ceases at about the time that generative cell division and pollen tube elongation begins.Boron addition enhances both ultraviolet and 4-nitroquinoline-1-oxide induced repair synthesis. By delaying the chemical mutagen initiation of repair until after germination has begun, we have been able to show that boron is most beneficial during the first hour of germination. It is postulated that this is achieved through an as yet unknown effect of boron on the supply of precursors before pollen cell metabolism is fully committed to pollen tube synthesis later in the germination period.