Over 1000 genes are involved in the DNA damage response of Escherichia coli

Changes in gene expression after treatment of Escherichia coli cultures with mitomycin C were assessed using gene array technology. Unexpectedly, a large number of genes (nearly 30% of all genes) displayed significant changes in their expression level. Analysis and classification of expression profiles of the corresponding genes allowed us to assign this large number of genes into one or two dozen small clusters of genes with similar expression profiles. This assignment allowed us to describe systematically the changes in the level of gene expression in response to DNA damage. Among the damage-induced genes, more than 100 are novel. From those genes involved in DNA metabolism that have not previously been shown to be induced by DNA damage, the mutS gene involved in mismatch repair is especially noteworthy. In addition to the SOS response, we observed the induction of other stress response pathways, such as those of oxidative stress and osmotic protection. Among the genes that are downregulated in response to DNA damage are numerous protein biosynthesis genes. Analysis of the gene expression data highlighted the essential involvement of sigma(s)-regulated genes and the general stress response network in the response to DNA damage.     

Identification of genes involved in terbinafine resistance in Aspergillus nidulans

AIMS: To determine the pattern and the genetic basis of resistance to terbinafine, a drug extensively used for the treatment of fungal infections in humans. METHODS AND RESULTS: Four resistant mutants from Aspergillus nidulans isolated after irradiation with ultraviolet light were crossed with the master strain F (MSF). Genetic analysis revealed that a single gene, located on chromosome IV, is responsible for resistance to terbinafine and that the alleles responsible for this resistance in these mutants are of a codominant or dominant nature at high terbinafine concentrations. Furthermore, the interaction of this mutation with another one identified on chromosome II causes the double mutant to be highly resistant. CONCLUSIONS: Periodic surveillance of antimycotic susceptibility would be an important measure in detecting the emergence and spread of resistance. Mutation in a single gene could be responsible for resistance to terbinafine and a genic interaction may be responsible for a higher level of antimycotic resistance. SIGNIFICANCE AND IMPACT OF THE STUDY: The understanding of the mechanisms that lead to changes in the sensitivity of a fungus to a given antifungal agent is important both in order to define strategies for the use of such agent and to guide the development of new antifungal agents.     

Tagging of genes involved in multidrug resistance in Aspergillus nidulans

We have used a plasmid containing the Neurospora crassa pyr4 gene to transform an Aspergillus nidulans pyrG89 mutant strain in the presence of BamHI, and isolated multidrug-sensitive mutants among the transformants. Using this approach, we hoped to identify genes whose products are important for drug resistance by analyzing gene disruptions that alter the drug sensitivity of the cell. About 1300 transformants isolated following transformation were screened for sensitivity to drugs or various stress agents with different and/or the same mechanism of action. Seventy-seven of these transformants showed sensitivity to at least one drug, while fourteen transformants showed a complex phenotype of sensitivity to different drugs. The pyr4 marker was shown to be tightly linked to the mutant phenotype in only 36% of the pleiotropic mutants analyzed in sexual crosses. Genetic crosses between our multidrug-sensitive transformants and cycloheximide-sensitive and imazalil-resistant mutants of A nidulans were performed to determine whether mutations were present at the same loci. We have shown that the gene imaD that confers resistance to imazalil may also be involved in cycloheximide and hygromycin sensitivity, since this mutation is allelic to scyB (mutant scy290). In addition, the cross between the transformant R223 and the imazalil-resistant mutant ima535 showed that both mutations are in the same complementation group, suggesting that the gene imaG could also be involved in cycloheximide and itraconazole sensitivity. Received: 30 August 1999 / Accepted: 22 February 2000     

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.     

Different roles of the Mre11 complex in the DNA damage response in Aspergillus nidulans

The Mre11-Rad50-Nbs1 protein complex has emerged as a central player in the cellular DNA damage response. Mutations in scaANBS1, which encodes the apparent homologue of human Nbs1 in Aspergillus nidulans, inhibit growth in the presence of the anti-topoisomerase I drug camptothecin. We have used the scaANBS1 cDNA as a bait in a yeast two-hybrid screening and report the identification of the A. nidulans Mre11 homologue (mreA). The inactivated mreA strain was more sensitive to several DNA damaging and oxidative stress agents. Septation in A. nidulans is dependent not only on the uvsBATR gene, but also on the mre11 complex. scaANBS1 and mreA genes are both involved in the DNA replication checkpoint whereas mreA is specifically involved in the intra-S-phase checkpoint. ScaANBS1 also participates in G2-M checkpoint control upon DNA damage caused by MMS. In addition, the scaANBS1 gene is also important for ascospore viability, whereas mreA is required for successful meiosis in A. nidulans. Consistent with this view, the Mre11 complex and the uvsCRAD51 gene are highly expressed at the mRNA level during the sexual development.     

Multiple catalase genes are differentially regulated in Aspergillus nidulans

Detoxification of hydrogen peroxide is a fundamental aspect of the cellular antioxidant responses in which catalases play a major role. Two differentially regulated catalase genes, catA and catB, have been studied in Aspergillus nidulans. Here we have characterized a third catalase gene, designated catC, which predicts a 475-amino-acid polypeptide containing a peroxisome-targeting signal. With a molecular mass of 54 kDa, CatC shows high similarity to other small-subunit monofunctional catalases and is most closely related to catalases from other fungi, Archaea, and animals. In contrast, the CatA (approximately 84 kDa) and CatB (approximately 79 kDa) enzymes belong to a family of large-subunit catalases, constituting a unique fungal and bacterial group. The catC gene displayed a relatively constant pattern of expression, not being induced by oxidative or other types of stress. Targeted disruption of catC eliminated a constitutive catalase activity not detected previously in zymogram gels. However, a catalase activity detected in catA catB mutant strains during late stationary phase was still present in catC and catABC null mutants, thus demonstrating the presence of a fourth catalase, here named catalase D (CatD). Neither catC nor catABC triple mutants showed any developmental defect, and both mutants grew as well as wild-type strains in H(2)O(2)-generating substrates, such as fatty acids, and/or purines as the sole carbon and nitrogen sources, respectively. CatD activity was induced during late stationary phase by glucose starvation, high temperature, and, to a lesser extent, H(2)O(2) treatment. The existence of at least four differentially regulated catalases indicates a large and regulated capability for H(2)O(2) detoxification in filamentous fungi.     

Two new genes involved in signalling ambient pH in Aspergillus nidulans

Two new genes, palH and palI, where mutations mimic the effects of acidic growth pH have been identified in Aspergillus nidulans. A palH mutation is phenotypically indistinguishable from mutations in the palA, palB, palC, and palF genes, whereas palI mutations differ only in that they allow some growth at pH 8. Mutations in palA, B, C, F, and H are epistatic to a palI mutation and the significance of this epistasis is discussed. Additionally, palE and palB mutations have been shown to be allelic. Thus, a total of six genes where mutations mimic acidic growth conditions has been identified.     

The uvsC and uvsE genes of Aspergillus nidulans are not required for the mutagenic repair of UV damage

The uvsC gene of Aspergillus nidulans is a homolog of the RAD51 gene of Saccharomyces cerevisiae. However, with respect to its effects on UV mutagenesis, it differs from the yeast gene, since it seems to be required for UV mutagenesis; however, this conclusion is based only on data from resting conidia. To further clarify the functional role of the uvsC gene, we tested the UV mutability of strains bearing a uvsC mutation in resting as well as in germinating conidia, by the p-fluoro-phenyl-alanine resistance test. We also evaluated the mutability of the uvsE mutant which belongs to the same epistatic group. Our results show that the uvsC and uvsE genes do not have a significant role in the mutagenic UV-repair pathway.     

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.     

The Aspergillus nidulans uvsB gene encodes an ATM-related kinase required for multiple facets of the DNA damage response

In Aspergillus nidulans, uvsB and uvsD belong to the same epistasis group of DNA repair mutants. Recent observations suggest that these genes are likely to control cell cycle checkpoint responses to DNA damage and incomplete replication. Consistent with this notion, we show here that UVSB is a member of the conserved family of ATM-related kinases. Phenotypic characterization of uvsB mutants shows that they possess defects in additional aspects of the DNA damage response besides checkpoint control, including inhibition of septum formation, regulation of gene expression, and induced mutagenesis. The musN227 mutation partially suppresses the poor growth and DNA damage sensitivity of uvsB mutants. Although musN227 partially suppresses several uvsB defects, it does not restore checkpoint function to uvsB mutants. Notably, the failure of uvsB mutants to restrain septum formation in the presence of DNA damage is suppressed by the musN227 mutation. We propose that UVSB functions as the central regulator of the A. nidulans DNA damage response, whereas MUSN promotes recovery by modulating a subset of the response.     

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.     

Two Aspergillus nidulans genes encoding methylenetetrahydrofolate reductases are up-regulated by homocysteine

Methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, a co-substrate in the synthesis of methionine from homocysteine. We have cloned and characterized two Aspergillus nidulans genes encoding MTHFRs: metA and metF. Mutations in either gene result in methionine requirement; the metA-encoded enzyme is responsible for only 10-15% of total MTHFR activity. These two enzymes belong to different classes of MTHFRs. Mutations in metA but not in the metF gene are suppressed by mutations resulting in enhancement of homocysteine synthesis. The expression of both genes is up-regulated by homocysteine.     

The Aspergillus niger acuA and acuB genes correspond to the facA and facB genes in Aspergillus nidulans

Mutants in Aspergillus niger unable to grow on acetate as a sole carbon source were previously isolated by resistance to 1.2% propionate medium containing 0.1% glucose. AcuA mutants lacked acetyl-CoA synthetase (ACS) activity and acuB mutants lacked both ACS and isocitrate lyase activity. An acuA mutant was transformed to the acu+ phenotype with a clone of ACS (facA) from Aspergillus nidulans. The acuB mutant was transformed with the A. niger facB clone which has been identified by cross-hybridisation of an A. nidulans facB clone. These results confirm that acuA in A. niger is the gene for ACS and acuB is analogous to the A. nidulans facB regulatory gene.     

5S rRNA genes from Aspergillus nidulans are not transcribed in Saccharomyces cerevisiae

Several different 5S rRNA genes from Aspergillus nidulans cloned in an Escherichia coli--Saccharomyces cerevisiae shuttle vector were introduced into S. cerevisiae cells by transformation. The A. nidulans 5S rRNA genes were not transcribed in S. cerevisiae.