Aspergillus nidulans as a model system to characterize the DNA damage response in eukaryotes

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.     

Transcriptome analysis of recombinant protein secretion by Aspergillus nidulans and the unfolded-protein response in vivo

Filamentous fungi have a high capacity for producing large amounts of secreted proteins, a property that has been exploited for commercial production of recombinant proteins. However, the secretory pathway, which is key to the production of extracellular proteins, is rather poorly characterized in filamentous fungi compared to yeast. We report the effects of recombinant protein secretion on gene expression levels in Aspergillus nidulans by directly comparing a bovine chymosin-producing strain with its parental wild-type strain in continuous culture by using expressed sequence tag microarrays. This approach demonstrated more subtle and specific changes in gene expression than those observed when mimicking the effects of protein overproduction by using a secretion blocker. The impact of overexpressing a secreted recombinant protein more closely resembles the unfolded-protein response in vivo.     

Regulation of septum formation in Aspergillus nidulans by a DNA damage checkpoint pathway.

In Aspergillus nidulans, germinating conidia undergo multiple rounds of nuclear division before the formation of the first septum. Previous characterization of temperature-sensitive sepB and sepJ mutations showed that although they block septation, they also cause moderate defects in chromosomal DNA metabolism. Results presented here demonstrate that a variety of other perturbations of chromosomal DNA metabolism also delay septum formation, suggesting that this is a general cellular response to the presence of sublethal DNA damage. Genetic evidence is provided that suggests that high levels of cyclin-dependent kinase (cdk) activity are required for septation in A. nidulans. Consistent with this notion, the inhibition of septum formation triggered by defects in chromosomal DNA metabolism depends upon Tyr-15 phosphorylation of the mitotic cdk p34nimX. Moreover, this response also requires elements of the DNA damage checkpoint pathway. A model is proposed that suggests that the DNA damage checkpoint response represents one of multiple sensory inputs that modulates p34nimX activity to control the timing of septum formation.     

Isolation of DNA from Aspergillus nidulans.

A procedure for isolation of DNA from Aspergillus nidulans on a preparative scale is described. Mechanical disruption of lyophilized material in high-salt medium and treatment with proteinase K, followed by sedimentation of the lysate into saturated CsC1 solution yielded pure, highly polymerized DNA.     

Repair of alkylation damage in the fungus Aspergillus nidulans

The repair of alkylation damage in Aspergillus nidulans was investigated. We have assayed soluble protein fractions for enzymes known to be involved in the repair of this type of damage in DNA. The presence of a glycosylase activity that can remove 3-methyladenine from DNA was demonstrated, as well as a DNA methyltransferase activity that appears to act against O6-methylguanine. In addition to this approach, a series of mutants were isolated which display increased sensitivity to alkylating agents (sag mutants). 5 such mutants were further characterized, and at least 4 are shown to map to genes which have not previously been characterized. The behaviour of double mutant combinations demonstrates the existence of at least 2 pathways for the repair of alkylation damage. The majority of the sag mutants (sagA1, sagB2, sag4 and sagE5) exhibit an increased sensitivity to a range of alkylating agents, but not to UV light, while sagC3, when irradiated at the germling stage, also shows sensitivity to UV. None of the mutants isolated are defective in either the 3-methyladenine DNA glycosylase activity, or the DNA methyltransferase activity, and the nature of the defects in these strains remains to be determined.     

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.     

Regulation of hyphal morphogenesis and the DNA damage response by the Aspergillus nidulans ATM homolog AtmA

Ataxia telangiectasia (A-T) is an inherited disorder characterized by progressive loss of motor function and susceptibility to cancer. The most prominent clinical feature observed in A-T patients is the degeneration of Purkinje motor neurons. Numerous studies have emphasized the role of the affected gene product, ATM, in the regulation of the DNA damage response. However, in Purkinje cells, the bulk of ATM localizes to the cytoplasm and may play a role in vesicle trafficking. The nature of this function, and its involvement in the pathology underlying A-T, remain unknown. Here we characterize the homolog of ATM (AtmA) in the filamentous fungus Aspergillus nidulans. In addition to its expected role in the DNA damage response, we find that AtmA is also required for polarized hyphal growth. We demonstrate that an atmA mutant fails to generate a stable axis of hyphal polarity. Notably, cytoplasmic microtubules display aberrant cortical interactions at the hyphal tip. Our results suggest that AtmA regulates the function and/or localization of landmark proteins required for the formation of a polarity axis. We propose that a similar function may contribute to the establishment of neuronal polarity.     

Survey of representative species of Aspergillus for regions of DNA homology to Aspergillus nidulans developmental genes

Summary This study surveyed five representative species of Aspergillus for regions of homology with previously cloned A. nidulans developmental genes. Areas of hybridization were found for all A. nidulans genes in the DNA of all of the Aspergillus species examined. All five species had a high level of homology with the tubC gene, but varied in degree of homology with the brlA gene and the SpoC1 gene cluster. These results suggest that DNA sequences analogous to A. nidulans developmental genes are found in other members of the genus and support the hypothesis that genetic investigations of A. nidulans could serve as model systems for genetic studies of other aspergilli.     

Genetic damage induced by ethyl alcohol in Aspergillus nidulans.

Heterozygous diploid conidia of Aspergillus nidulans were treated during germination with ethyl alcohol in concentrations ranging from 0.25% to 20% (v/v). The diploid strain carried three recessive conidial color mutations, in addition to genetic markers on all eight pairs of linkage groups. It was thereby possible to detect events of crossing over, non-disjunction, and mutation. An increase in the dose of ethanol was associated with a decrease in conidial viability and an increase in the relative and absolute frequencies of formation of (a) normal colonies which produced colored sectors and (b) phenotypically abnormal colonies, the majority of which (83.1%) produced normal sectors. At a concentration of 5% (v/v) ethanol, the survivors included 17.59% of the former and 44.7% of the latter colonies. Genetic analysis of the various segregants suggested that the frequencies of both mitotic crossing over and non-disjunction or the misdistribution of chromosomes were increased by ethanol. Among 133 abnormal colonies which segregated normal clones, 79 (59.4%) were associated with one of these genetic events. A total of 297 haploids and 130 diploids arose as normal segregants from the abnormal colonies. There were 31 recognizable events of non-disjunction and 14 crossing over in linkage groups I and II, where these events could be distinguished. These data suggested that the predominant effect of ethanol was a disruption of chromosome distribution. A cytological examination of ethanol-treated, germinating conidia revealed an interference with the mitotic spindle apparatus. The frequency of detectable spindles decreased more than 3-fold after 8 h exposure to 5% (v/v) ethanol. This finding supported the conclusion that ethanol disrupted chromosome distribution, and suggested the mechanism by which it does so. Human clinical data on alcohol consumption were examined in light of these findings.     

Temperature-shift analysis of conidial development in Aspergillus nidulans

Temperature-shift experiments have been performed on spore-originated colonies of 11 thermosensitive aconidial mutants of Aspergillus nidulans in order to determine the latest time of shift to the restrictive temperature that prevents the initiation of conidiation. This time defines the beginning of the thermosensitive period (TSP) of the mutant. Eight of the mutants have TSPs that begin in the 7-hour period (32–39 hr) just prior to the first appearance of conidia-bearing structures, while 3 of the mutants have TSPs that begin later and very close to the time of onset of conidiation (45 hr). Thus no mutant of the set has a TSP that begins during the first 32 hr of vegetative growth of spore-originated colonies. For all mutants, an upshift performed after the beginning of the TSP allows initiation of conidiation at close to the normal time and at the normal rate, but results in an abrupt cessation of conidiation at some fixed time after upshift, characteristic of the mutant. The mutant whose TSP begins the earliest (aco-49) is exceptional in that, if conidiation is suppressed by growth of colonies in submerged culture, this mutant becomes thermoinsensitive during vegetative submerged growth; in contrast, the remaining 10 mutants become thermoinsensitive only after the suppressive condition has been relieved. We discuss the possibility that this exceptional mutant is defective in a function required for initiation of the process that ultimately results in the formation of conidia.     

Genetic interactions of the Aspergillus nidulans atmAATM homolog with different components of the DNA damage response pathway

Ataxia telangiectasia mutated (ATM) is a phosphatidyl-3-kinase-related protein kinase that functions as a central regulator of the DNA damage response in eukaryotic cells. In humans, mutations in ATM cause the devastating neurodegenerative disease ataxia telangiectasia. Previously, we characterized the homolog of ATM (AtmA) in the filamentous fungus Aspergillus nidulans. In addition to its expected role in the DNA damage response, we found that AtmA is also required for polarized hyphal growth. Here, we extended these studies by investigating which components of the DNA damage response pathway are interacting with AtmA. The AtmA(ATM) loss of function caused synthetic lethality when combined with mutation in UvsB(ATR). Our results suggest that AtmA and UvsB are interacting and they are probably partially redundant in terms of DNA damage sensing and/or repairing and polar growth. We identified and inactivated A. nidulans chkA(CHK1) and chkB(CHK2) genes. These genes are also redundantly involved in A. nidulans DNA damage response. We constructed several combinations of double mutants for DeltaatmA, DeltauvsB, DeltachkA, and DeltachkB. We observed a complex genetic relationship with these mutations during the DNA replication checkpoint and DNA damage response. Finally, we observed epistatic and synergistic interactions between AtmA, and bimE(APC1), ankA(WEE1) and the cdc2-related kinase npkA, at S-phase checkpoint and in response to DNA-damaging agents.     

The genetic analysis of mitosis in Aspergillus nidulans

We describe here recent work on the molecular genetics of mitosis in the filamentous fungus Aspergillus nidulans. Aspergillus is one of three simple eukaryotes with powerful genetic systems that have been used to analyze mitosis. The modern molecular biological techniques available with this organism have made it possible to use mutations to identify genes and proteins that play an important role in mitosis. Three Aspergillus genes that affect mitosis are described. One gene, nimA, is specifically expressed late in the cell cycle and codes for a putative protein kinase that induces mitosis, even in cells blocked in S-phase. The second gene, bimG, codes for a putative phosphatase that interacts functionally with the nimA kinase. The third gene, bimE, codes for a protein that suppresses mitosis during interphase, apparently by keeping nimA turned off. None of these genes appear to be similar to any of the genes affecting mitosis that have been characterized in other eukaryotes, but rather appear to be elements of a system that prevents mitosis from occurring during interphase.     

Genetic analysis of Aspergillus nidulans AmdS+ transformants

Summary To correlate the genetic background of the Aspergillus nidulans amdS deletion strain MH1277 with the integrational behaviour of transforming vectors, classical genetic methods were used to construct AmdS^- strains in which whole chromosomes had been exchanged with those of a master strain. Progeny strains were transformed to the AmdS⁺ phenotype with vector p3SR2. From Southern analysis it was concluded that transformants from all constructions contained tandemly repeated, multiple copy inserts of vector DNA as found for MH1277-derived AmdS⁺ transformants.AmdS⁺ transformants of MH1277 were analysed genetically to prove that the transformant phenotype is genome linked and that transformation by integration can take place on various chromosomes. In one case the AmdS⁺ property showed linkage to both chromosomes II and IV, due to a chromosomal translocation. Sexual analysis of two transformants with AmdS⁺ insertions on the same chromosome revealed a considerable instability of the AmdS⁺ phenotype in one of the strains upon selfing. Due to this instability no decisive answer could be given for the degree of linkage between the AmdS⁺ insertions in these transformants.     

DNA damage in cells exposed to ionizing radiation

Ionizing radiation induces variety of structural lesions in DNA of irradiated organisms. Their formation depends largely on the degree of cell oxygenation, the level of endogenous antioxidants, on DNA-protein complexes and compactization of DNA in the chromatin and activity of DNA repair systems. All ionizing radiation-induced DNA lesions can arbitrarily be divided into two groups. Group 1 includes singly damaged sites (single-sites): base modification, single-strand breaks, alkaline-labile sites (including a basic sites). Group 2 contains: locally multiply damaged sites (clustered lesions), double-strand breaks, intermolecular cross-links. The yields of lesions of group 2 increases with high linear energy transfer of radiation and these lesions play a dominant role in the radiation death, formation of chromosome and gene mutations, cell transformation.     

Sensitivity to camptothecin in Aspergillus nidulans identifies a novel gene, scaA + , related to the cellular DNA damage response

The anti-cancer drug camptothecin targets eukaryotic DNA topoisomerase I by trapping the covalent complex formed between the catalytically active enzyme and DNA. We are interested in identifying factors, other than topoisomerase I, that are involved in mediating cellular sensitivity to camptothecin. To this end, we have isolated eighteen mutants that are sensitive to camptothecin (sca) in the filamentous fungus Aspergillus nidulans and characterised one of them, sca299. The mutant sca299 is hypersensitive to camptothecin, and sensitive to several different mutagenic agents and to actinomycin D. Using temperature-sensitive mutations in genes that are known to regulate the cell cycle, we showed that the camptothecin sensitivity of the mutant sca299 is not affected by a mitotic block. The abnormal nuclear morphology observed in the sca299 mutant strain suggests that the germlings might be undergoing mitosis in the presence of unrepaired DNA damage, which would result in mitotic catastrophe. The hypersensitivity of the sca299 mutant to camptothecin does not result from elevated levels of topoisomerase I mRNA or from alterations in enzyme activity. Using DNA-mediated complementation of the sca299 mutant phenotype, the scaA+ gene was cloned. This gene encodes a 594-amino acid product; moderate structural similarity suggests that the scaA gene product may be related to the human nibrin gene which encodes a product involved in DNA double-strand break repair. Strains disrupted in the scaA gene were sensitive to the anti-topoisomerase I agent berberine, the DNA crosslinking agents mitomycin C and cis-platinum, and also to t-butyl hydroperoxide, which is an inducer of oxidative stress.     

Endo-exonuclease 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.