uvsF201 was the first highly UV-sensitive repair-defective mutation isolated in Aspergillus nidulans. It showed epistasis only with postreplication repair mutations, but caused lethal interactions with many other repair-defective strains. Unexpectedly, closest homology of uvsF was found to the large subunit of human DNA replication factor RFC that is essential for DNA replication. Sequencing of the uvsF201 region identified changes at two close base pairs and the corresponding amino acids in the 5′-region of uvsFRFC1. This viable mutant represents a novel and possibly important type. Additional sequencing of the uvsF region confirmed a mitochondrial ribosomal protein gene, mrpAL16, closely adjacent, head-to-head with a 0.2kb joint promoter region. MMS-induced transcription of both the genes, but especially uvsFRFC1, providing evidence for a function in DNA damage response.

Interest in DNA repair in Aspergillus nidulans had mainly grown out of studies of three different biological processes, namely mitotic recombination, inducible responses to detrimental environmental changes, and genetic control of the cell cycle. Ron Morris started the investigation of the genetic control of the cell cycle by screening hundreds of cell cycle temperature sensitive Aspergillus mutants. The sequencing and innovative analysis of these genes revealed not only several components of the cell cycle machinery that are directly involved in checkpoint response, but also components required for DNA replication and DNA damage response machinery. Here, we will provide an overview about currently known aspects of the DNA damage response in A. nidulans. Emphasis is put on analyzed mutants that are available and review epistatic relationships and other interactions among them. Furthermore, a comprehensive list of A. nidulans genes involved in different processes of the DNA damage response, as identified by homology of genome sequences with well-characterized human and yeast DNA repair genes, is shown.

BIMD of Aspergillus nidulans belongs to a highly conserved protein family implicated, in filamentous fungi, in sister-chromatid cohesion and DNA repair. We show here that BIMD is chromosome associated at all stages, except from late prophase through anaphase, during mitosis and meiosis, and is involved in several aspects of both programs. First, bimD + function must be executed during S through M. Second, in bimD6 germlings, mitotic nuclear divisions and overall cellular program occur more rapidly than in wild type. Thus, BIMD, an abundant chromosomal protein, is a negative regulator of normal cell cycle progression. Third, bimD6 reduces the level of mitotic interhomolog recombination but does not alter the ratio between crossover and noncrossover outcomes. Moreover, bimD6 is normal for intrachromosomal recombination. Therefore, BIMD is probably not involved in the enzymology of recombinational repair per se . Finally, during meiosis, staining of the Sordaria ortholog Spo76p delineates robust chromosomal axes, whereas BIMD stains all chromatin. SPO76 and bimD are functional homologs with respect to their roles in mitotic chromosome metabolism but not in meiosis. We propose that BIMD exerts its diverse influences on cell cycle progression as well as chromosome morphogenesis and recombination by modulating chromosome structure.

The UV-sensitive mutant uvsF201 of Aspergillus nidulans shows increased spontaneous and UV-induced mutation and generally resembles mutants defective in nucleotide excision repair (NER). Fully-complementing uvsF clones were isolated from cosmid and cDNA libraries for sequencing. The uvsF gene is approximately 3.75 kb long and codes for a predicted polypeptide of 1092 amino acids (aa). Three small introns are clustered early in the coding region of the protein. A major part of the sequence shows homology to human, mouse and yeast RFC1 genes which code for the large subunit of the DNA replication factor C. The uvsF gene product may therefore function primarily in general DNA replication but in addition be required for the replication step of DNA repair. Extended sequencing of the uvsF gene region identified a second closely adjacent gene of unknown function which is divergently transcribed from a small (0.2 kb) intergenic promoter region. © 1997 Elsevier Science B.V.

Two genes of Aspergillus nidulans are known to function in UV mutagenesis, but have been assigned to different epistasis groups: uvsC, which is also required for meiosis and mitotic recombination, and uvsI, which may have no other function. To clarify their role in error-prone repair and to investigate their interaction, uvsI and uvsC single and uvsI;uvsC double mutant strains were further tested for mutagen sensitivities and characterized for effects on mutation. Spontaneous and induced frequencies were compared in forward and reverse mutation assays. All results confirmed that uvsI and uvsC are members of different epistasis groups, and demonstrated that these uvs mutants have very different defects in UV mutagenesis. The uvsI strains showed wild-type frequencies in all forward mutation tests, but greatly reduced spontaneous and UV-induced reversion of some, but not other, point mutations. In contrast, uvsC had similar effects in all assay systems: namely pronounced mutator effects and greatly reduced UV mutagenesis. Interestingly, the uvsI;uvsC double mutant strains differed from both single mutants; they clearly showed synergism for all types of reversion tested: none were ever obtained spontaneously, nor after induction by UV or EMS (ethylmethane sulfonate). Based on these results, we conclude that uvsI is active in a mutation-specific, specialized error-prone repair process in Aspergillus. In contrast, uvsC, which is now known to show sequence homology to recA, has a basic function in mutagenic UV repair in addition to recombinational repair, similar to recA of Escherichia coli.

We cloned a DNA repair gene, mus-8, of Neurospora crassa and sequenced the genomic DNA and cDNA. Nucleotide-sequence analysis indicated that the mus-8 gene contains an open reading frame (ORF) of 456 bp, interrupted by three small introns. The deduced amino-acid sequence showed that the mus-8 gene encodes a 17 kDa protein which has 77.5% and 83.3% identity to the Rad6 protein of Saccharomyces cerevisiae and the rhp6+ protein of Schizosaccharomyces pombe, respectively. The Rad6 protein is a ubiquitin-conjugating enzyme (E2) and is required for DNA repair, mutagenesis, and sporulation in yeast. Introduction of the mus-8 gene into a S. cerevisiae rad6 mutant resulted in significant recovery of DNA repair functions, especially UV-mutagenesis, and also sporulation, both of which are defective in the rad6 mutant. It is therefore postulated that mus-8 of Neurospora has a function very similar to that demonstrated for RAD6 of S. cerevisiae.

Genetic analysis recently identified nine new DNA repair genes of Aspergillus nidulans, musK - musS, which map on several different chromosomes (Käfer 1994, preceding article in this Newsletter). Such mus mutants are sensitive to certain chemical mutagens, but not sensitive or only slightly sensitive to UV and gamma-radiation. To identify epistatic interactions with members of the 4 Uvs groups, double mutants strains were isolated (Käfer and Chae 1994 Curr. Genet. 25:223-232). However, some mus;uvs double mutants could not be analyzed because they grew too poorly or were lethal (showing "synthetic enhancement in gene interaction"; Guarente 1993 Trends Genet. 9:362-366). The opposite effect was also found; namely interaction which led to improved recovery and growth (or "rescue") as documented here.

With the development of molecular biology techniques and their application to the analysis of cellular events, the isolation of total nucleic acids in Aspergillus nidulans for Southern and Northern hybridization has become routine.

Mutation in the bimD gene of Aspergillus nidulans results in a mitotic block in anaphase characterized by a defective mitosis. Mutation in bimD also confers, at temperatures permissive for the mitotic arrest phenotype, an increased sensitivity to DNA damaging agents, including methyl methanesulfonate and ultraviolet light. In order to better understand the relationship between DNA damage and mitotic progression, we cloned the bimD gene from Aspergillus. A cosmid containing the bimD gene was identified among pools of cosmids by cotransformation with the nutritional selective pyrG gene of a strain carrying the recessive, temperature-sensitive lethal bimD6 mutation. The bimD gene encodes a predicted polypeptide of 166,000 daltons in mass and contains amino acid sequence motifs similar to those found in some DNA-binding transcription factors. These sequences include a basic domain followed by a leucine zipper, which together are called a bZIP motif, and a carboxyl-terminal domain enriched in acidic amino acids. Overexpression of the wild-type bimD protein resulted in an arrest of the nuclear division cycle that was reversible and determined to be in either the G1 or S phase of the cell cycle. Our data suggest that bimD may play an essential regulatory role relating to DNA metabolism which is required for a successful mitosis.

Methyl methane-sulfonate (MMS)-sensitive, radiation-induced mutants of Aspergillus were shown to define nine new DNA repair genes, musK to musS. To test mus mutations for effects on mitotic recombination, intergenic crossing over was assayed between color markers and their centromeres, and intragenic recombination between two distinguishable adE alleles. Of eight mutants analyzed, four showed significant deviations from mus+ controls in both tests. Two mutations, musK and musL, reduced recombination, while musN and musQ caused increases. In contrast, musO diploids produced significantly higher levels only for intragenic recombination. Effects were relatively small, but averages between hypo- and hyperrec mus differed 15-20-fold. In musL diploids, most of the rare color segregants resulted from mitotic malsegregation rather than intergenic crossing over. This indicates that the musL gene product is required for recombination and that DNA lesions lead to chromosome loss when it is deficient. In addition, analysis of the genotypes of intragenic (ad+) recombinants showed that the musL mutation specifically reduced single allele conversion but increased complex conversion types (especially recombinants homozygous for ad+). Similar analysis revealed differences between the effects of two hyperrec mutations; musN apparently caused high levels solely of mitotic crossing over, while musQ increased various conversion types but not reciprocal crossovers. These results suggest that mitotic gene conversion and crossing over, while generally associated, are affected differentially in some of the mus strains of Aspergillus nidulans.

Endo-exonuclease (EE) has been found in both active and inactive, but trypsin-activatable, forms in Aspergillus nidulans. Active EE was present mainly in nuclei, mitochondria, and vacuoles, while trypsin-activatable EE was mainly in the cytosol. The active form accounts for over 90% of the neutral deoxyribonuclease activity extracted from mycelia. A single strand (ss) DNA-binding EE associated with a 28 kilodalton (kDa) polypeptide was partially purified and characterized. It was found to closely resemble, in size and enzymological properties, the ss-DNA-binding EE previously purified from Neurospora crassa. Aspergillus nidulans EE was also found to be immunochemically related to the N. crassa EE and, like that enzyme, was probably derived from a polypeptide of 90 kDa or larger through proteolysis during extraction and purification. It had divalent metal ion-dependent (Mg2+, Mn2+, or Zn2+) activity on both DNA and RNA, which ultimately produced small 5'-P-terminated oligonucleotides. The nuclease activity was mixed endo- and exo-nucleolytic with ss-DNA as substrate, but largely exonucleolytic with double strand (ds) DNA. Superhelical phi X-174 DNA was nicked by EE to form relaxed circular and then linear ds-DNA, which was rapidly degraded to shorter fragments. Linearized pBR322 DNA was extensively nicked internally under conditions where there was relatively low exonuclease activity, but this nicking required that 5'-P-termini be present on the linear ds-DNA. The levels of active EE found in extracts of two recombination-deficient mutants of A. nidulans, uvsC and uvsE, dit not differ significantly from those in extracts of the wild type.

High levels of nuclease activities were identified in filtrates ofAspergillus cultures after growth in low- but not in high-phosphate media. Deoxyribonuclease activities, characterized extensively by column chromatography, showed a coincident single peak for ss- and ds-DNase which was distinct from the peak for RNase. Both ss-DNase and ds-DNase are endonucleolytic and showed the highest activity in the presence of Ca2+ and Mn2+ (atpH 8.0). They also showed identical heat sensitivities suggesting that a single, phosphate-repressible DNase was secreted. This enzyme, therefore, corresponds to the well-characterized extracellular DNase A ofNeurospora. However, theAspergillus DNase A did not cross-react with antisera to secretedNeurospora nucleases and showed different chromatographic properties, and active peptides of different sizes were visualized on DNA activity gels. The increasing derepression ofAspergillus DNase A by decreasing phosphate levels was similar to that of secreted alkaline phosphatase and these increases were both abolished by the regulatory mutantpalcA.

Mutations were induced in Neurospora which cause increased sensitivity to MMS (methyl methane-sulfonate) and other mutagens. Genetic analysis of such mus demonstrated that some of them defined new DNA repair genes (mus-21, and mus-27 to mus-30), while others represented new alleles in previously known genes. To characterize them further, and especially to identify rec- types which have not yet been found in this species, many MMS-sensitive strains were tested for cross-sensitivities to bleomycin (BLM) and to hydrogen peroxide (H2O2) to which some rec- of other species are hypersensitive. In Neurospora, many of the MMS-sensitive mutants were found to be cross-sensitive to BLM and frequently these were also hypersensitive to ionizing radiation. Bleomycin sensitivity was demonstrated for all alleles of 10 different genes, 4 of them new ones, with mus-27 being the most sensitive of the latter (resembling uvs-6; Koga and Schroeder, 1987, Mutation Res., 183, 139). In contrast, very few of the MMS-sensitive mutants were hypersensitive to H2O2 and, in general, results of H2O2 tests were variable and differences between strains small. However, consistent deviations from wild type were observed in a few cases (most clearly for mus-9 and mus-11) when results from treatments of germinating conidia were compared with those of non-growing ones.

Abstract The duplication Dp(D305) is shown to cover the erg-3 locus (which encodes the ergosterol biosynthetic enzyme C-14 reductase). Additionally the efficiency of RIP in Dp(D305) is shown to be very low. This low efficiency may be due to the marked instability of the duplication in the premeiotic stage of the sexual cross. Premeiotic instability might also account for the low frequency with which duplication progeny are recovered from Dp(D305) x Normal crosses.

Some time ago, a number of mutants hypersensitive to MMS (methyl methane-sulfonate) were induced in Neurospora to obtain further types of DNA repair-deficient mutants; e.g., rec^- types not yet identified in Neurospora (meiotic-defective mutants generally are "hyperrec"; Schroeder 1986 Curr. Genet. 10:381-387).

Information of the genetic location of pyrG and its growth response under various conditions has become important with the recent cloning of pyrG (Oakley et al. 1987. Gene 61:385-399) and with the use of pyrG strains as recipients for transformation when pyr-4 cloning vectors are used for A. nidulans libraries (e.g. May et al. 1985 J. Cell Biol. 101:712-719 ; Osmani et al. 1987 J. Cell Biol. 104:1495-1504).

The possibility of more than 1 target for genotoxic effects of methyl methanesulphonate (MMS) was investigated, using mitotic test systems of the fungus Aspergillus. Haploid and diploid strains were exposed, either as dormant conidia or during mitosis, and analysed for induced aneuploidy and effects on genetic segregation. MMS treatment of haploid strains resulted in dose-dependent increases of stable mutants with altered phenotypes and semi-stable unbalanced aberrations (presumably duplications). In addition, but only in dividing cells, MMS induced unstable aneuploids. These mostly were hyperhaploid with few extra chromosomes and could be identified by comparison with standard disomic phenotypes. When well-marked diploids were treated 3 types of effect could be distinguished, using genetic and phenotypic criteria: (1) Clastogenic and mutagenic effects which caused dose-dependent increases of partial aneuploids with various abnormal phenotypes. These showed secondary genetic segregation of all types and produced euploid normal sectors by eliminating damaged chromosome segments. In addition, but only in dividing nuclei, MMS induced 2 types of segregation: (2) Reciprocal crossing-over at high frequency, recognisable as half or quarter colonies of mutant colour and in some cases as 'twin spots' (i.e., complementary pairs); (3) Trisomics and other aneuploids which showed characteristic phenotypes and expected segregation of markers: the types recovered indicate random malsegregation of chromosomes (occasional deviations resulted from coincidence with induced crossing-over). These results suggest that MMS may have 2 (or more) targets for genotoxic effects: DNA, as evident from induced mutations and aberrations, and from induced recombination in dividing cells; some non-DNA target (nucleotide or protein) essential for nuclear division and susceptible to alkylation, resulting in malsegregation and primary aneuploidy.

All available amino acid-requiring mutants of Aspergillus nidulans were found to be hypersensitive to MMS (methyl methanesulfonate) to various degrees. On MMS media, secondary mutations could be selected which suppress this MMS sensitivity but do not affect the requirement. Many such mutations were analyzed and found to be alleles of one gene, smsA (= suppressor of MMS sensitivity), which mapped distal on the right arm of chromosome V. This gene is more likely to be involved in general regulation of amino acid biosynthesis than MMS uptake, since a variety of pathway interactions were clearly modified by smsA suppressors in the absence of MMS.

In Aspergillus several types of test systems have been developed for detection of chemicals which induce aneuploidy and/or malsegregation of chromosomes. Results from 23 papers were reviewed in which numerical data for 42 chemicals had been reported. The test systems fall into two groups. One group includes all purely genetic tests that detect euploid mitotic segregants from heterozygous diploids and identify these either as products of malsegregation of chromosomes or as products of crossing-over (13 papers, several reviewed in detail previously; Käfer et al. (1982) and Scott et al. (1982)). The other group includes tests that treat haploid or diploid strains and detect aneuploids as unstable abnormally growing segregants which can be identified as specific disomics or trisomics by their characteristic phenotypes. In addition, such tests characterize abnormal segregants from heterozygous diploids by correlating phenotypes with patterns of genetic segregation in spontaneous euploid sectors. This analysis makes it possible to distinguish between induced primary aneuploidy of whole chromosomes and partial tri- or monosomy resulting from chromosome breakage and secondary spontaneous malsegregation (10 papers). Based on results of both types of tests, it is postulated that chemicals which cause increases of euploid malsegregants, but not of crossovers, normally induce aneuploids as primary products (as shown for 7 of the 14 cases). These include compounds which damage spindles or membranes (especially the well-known haploidizing agents) and generally are effective only when growing cells are exposed. (8 chemicals that may belong in this category could not be classified for certain, because information was insufficient.) On the other hand, chemicals which cause increases of all types of euploid segregants (11 cases), mostly induce drastic mutations and aberrations as primary effects and cause spontaneous malsegregation or crossing-over only as secondary events (as demonstrated for radiation-induced abnormals). In addition, a few chemicals were negative, because they increased only crossing-over or showed no increased segregation at all at concentrations which reduced survival or growth rate (9 cases). Recommendations are made for standardization of methods and protocols. New tester strains and specific procedures are outlined which should be useful for conclusive tests of chemicals that may induce aneuploidy.