Transcriptome Analysis of Aspergillus nidulans Exposed to Camptothecin-Induced DNA Damage

ABSTRACT We have used an Aspergillus nidulans macroarray carrying sequences of 2,787 genes from this fungus to monitor gene expression of both wild-type and uvsBATR (the homologue of the ATR gene) deletion mutant strains in a time course exposure to camptothecin (CPT). The results revealed a total of 1,512 and 1,700 genes in the wild-type and uvsBATR deletion mutant strains that displayed a statistically significant difference at at least one experimental time point. We characterized six genes that have increased mRNA expression in the presence of CPT in the wild-type strain relative to the uvsBATR mutant strain: fhdA (encoding a forkhead-associated domain protein), tprA (encoding a hypothetical protein that contains a tetratrico peptide repeat), mshA (encoding a MutS homologue involved in mismatch repair), phbA (encoding a prohibitin homologue), uvsCRAD51 (the homologue of the RAD51 gene), and cshA (encoding a homologue of the excision repair protein ERCC-6 [Cockayne's syndrome protein]). The induced transcript levels of these genes in the presence of CPT require uvsBATR. These genes were deleted, and surprisingly, only the ΔuvsC mutant strain was sensitive to CPT; however, the others displayed sensitivity to a range of DNA-damaging and oxidative stress agents. These results indicate that the selected genes when inactivated display very complex and heterogeneous sensitivity behavior during growth in the presence of agents that directly or indirectly cause DNA damage. Moreover, with the exception of UvsC, deletion of each of these genes partially suppressed the sensitivity of the ΔuvsB strain to menadione and paraquat. Our results provide the first insight into the overall complexity of the response to DNA damage in filamentous fungi and suggest that multiple pathways may act in parallel to mediate DNA repair.

[1]  E. Käfer.  Meiotic and mitotic recombination in Aspergillus and its chromosomal aberrations. , 1977, Advances in genetics.

[2]  N. Morris,et al.  Kinetics of the nuclear division cycle of Aspergillus nidulans , 1983, Journal of bacteriology.

[3]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[4]  S. Osmani,et al.  Regulation of the mRNA levels of nimA, a gene required for the G2-M transition in Aspergillus nidulans , 1987, The Journal of cell biology.

[5]  L. Liu,et al.  DNA topoisomerase poisons as antitumor drugs. , 1989, Annual review of biochemistry.

[6]  C. Yu,et al.  Camptothecin overcomes MDR1-mediated resistance in human KB carcinoma cells. , 1991, Cancer research.

[7]  Leroy F. Liu.  DNA Topoisomerases : Topoisomerase-targeting drugs , 1994 .

[8]  N. Osheroff,et al.  Topoisomerase Poisons: Harnessing the Dark Side of Enzyme Mechanism (*) , 1995, The Journal of Biological Chemistry.

[9]  Y. Pommier,et al.  Eukaryotic DNA topoisomerases I. , 1995, Biochimica et biophysica acta.

[10]  O. White,et al.  TDB: new databases for biological discovery. , 1996, Methods in enzymology.

[11]  Thomas L. Madden,et al.  Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. , 1997, Nucleic acids research.

[12]  Y. Shiloh,et al.  Ataxia-telangiectasia and the Nijmegen breakage syndrome: related disorders but genes apart. , 1997, Annual review of genetics.

[13]  T. Uchiumi,et al.  A canalicular multispecific organic anion transporter (cMOAT) antisense cDNA enhances drug sensitivity in human hepatic cancer cells. , 1997, Cancer research.

[14]  M. Bjornsti,et al.  Camptothecin Sensitivity Is Mediated by the Pleiotropic Drug Resistance Network in Yeast* , 1997, The Journal of Biological Chemistry.

[15]  Y. Shiloh,et al.  ATM: from gene to function. , 1998, Human molecular genetics.

[16]  J. Berger.  Structure of DNA topoisomerases. , 1998, Biochimica et biophysica acta.

[17]  P Green,et al.  Base-calling of automated sequencer traces using phred. II. Error probabilities. , 1998, Genome research.

[18]  T. DeWeese,et al.  Mouse embryonic stem cells carrying one or two defective Msh2 alleles respond abnormally to oxidative stress inflicted by low-level radiation. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[19]  M. Earley,et al.  The role of mismatch repair in the prevention of base pair mutations in Saccharomyces cerevisiae. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[20]  P. Green,et al.  Base-calling of automated sequencer traces using phred. I. Accuracy assessment. , 1998, Genome research.

[21]  P. Green,et al.  Consed: a graphical tool for sequence finishing. , 1998, Genome research.

[22]  S. Osmani,et al.  Checkpoint defects leading to premature mitosis also cause endoreplication of DNA in Aspergillus nidulans. , 1999, Molecular biology of the cell.

[23]  Roger E Bumgarner,et al.  Comparative hybridization of an array of 21,500 ovarian cDNAs for the discovery of genes overexpressed in ovarian carcinomas. , 1999, Gene.

[24]  L. Hillier,et al.  High-throughput plasmid DNA purification for 3 cents per sample. , 1999, Nucleic acids research.

[25]  G. Marsischky,et al.  MSH2 and MSH6 are required for removal of adenine misincorporated opposite 8-oxo-guanine in S. cerevisiae. , 1999, Molecular cell.

[26]  X. Huang,et al.  CAP3: A DNA sequence assembly program. , 1999, Genome research.

[27]  C. d’Enfert,et al.  A rapid method for efficient gene replacement in the filamentous fungus Aspergillus nidulans. , 2000, Nucleic acids research.

[28]  S. Elledge,et al.  The DNA damage response: putting checkpoints in perspective , 2000, Nature.

[29]  G. Goldman,et al.  Sensitivity to camptothecin in Aspergillus nidulans identifies a novel gene, scaA+, related to the cellular DNA damage response , 2001, Molecular Genetics and Genomics.

[30]  R. Abraham.  Cell cycle checkpoint signaling through the ATM and ATR kinases. , 2001, Genes & development.

[31]  Y. Itoh,et al.  An Aspergillus nidulans uvsC null mutant is deficient in homologous DNA integration , 2001, Molecular and General Genetics MGG.

[32]  T. E. Wilson,et al.  Yeast Tdp1 and Rad1-Rad10 function as redundant pathways for repairing Top1 replicative damage , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[33]  Chunyan Liu,et al.  Repair of topoisomerase I covalent complexes in the absence of the tyrosyl-DNA phosphodiesterase Tdp1 , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[34]  Richard Fishel,et al.  Activation of Human MutS Homologs by 8-Oxo-guanine DNA Damage* , 2002, The Journal of Biological Chemistry.

[35]  S. Elledge,et al.  Checking on the fork: the DNA-replication stress-response pathway. , 2002, Trends in cell biology.

[36]  Ronald W. Davis,et al.  Transcriptional response of Saccharomyces cerevisiae to DNA-damaging agents does not identify the genes that protect against these agents , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[37]  W. Cliby,et al.  S Phase and G2 Arrests Induced by Topoisomerase I Poisons Are Dependent on ATR Kinase Function* , 2002, The Journal of Biological Chemistry.

[38]  D. Chang,et al.  Oxidative stress inactivates the human DNA mismatch repair system. , 2002, American journal of physiology. Cell physiology.

[39]  William C Reinhold,et al.  Transcriptional regulation of mitotic genes by camptothecin-induced DNA damage: microarray analysis of dose- and time-dependent effects. , 2002, Cancer research.

[40]  C. P. Semighini,et al.  Quantitative Analysis of the Relative Transcript Levels of ABC Transporter Atr Genes in Aspergillus nidulans by Real-Time Reverse Transcription-PCR Assay , 2002, Applied and Environmental Microbiology.

[41]  T. Weinert,et al.  Toward maintaining the genome: DNA damage and replication checkpoints. , 2002, Annual review of genetics.

[42]  Hideko Urushihara,et al.  PCR-mediated generation of a gene disruption construct without the use of DNA ligase and plasmid vectors. , 2002, Nucleic acids research.

[43]  G. Goldman,et al.  Identification of a topoisomerase I mutant, scsA1, as an extragenic suppressor of a mutation in scaA(NBS1), the apparent homolog of human nibrin in Aspergillus nidulans. , 2003, Genetics.

[44]  J. Hoeijmakers,et al.  Cell type-specific hypersensitivity to oxidative damage in CSB and XPA mice. , 2003, DNA repair.

[45]  J. Qin,et al.  Molecular Anatomy of the DNA Damage and Replication Checkpoints , 2003, Radiation research.

[46]  John N Weinstein,et al.  Impact of p53 knockout and topotecan treatment on gene expression profiles in human colon carcinoma cells: a pharmacogenomic study. , 2003, Cancer research.

[47]  J. Qin,et al.  MSH2 and ATR form a signaling module and regulate two branches of the damage response to DNA methylation , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[48]  Y.-M. Zhang,et al.  The DNA excision repair system of the highly radioresistant bacterium Deinococcus radiodurans is facilitated by the pentose phosphate pathway , 2003, Molecular microbiology.

[49]  E. Rubin,et al.  Mechanisms of resistance to topoisomerase I-targeting drugs , 2003, Oncogene.

[50]  Yingnian Yu,et al.  ATM, ATR and DNA-PK: initiators of the cellular genotoxic stress responses. , 2003, Carcinogenesis.

[51]  S. Chellappan,et al.  A putative coiled-coil domain of prohibitin is sufficient to repress E2F1-mediated transcription and induce apoptosis. , 2003, Biochemical and biophysical research communications.

[52]  C. P. Semighini,et al.  Different roles of the Mre11 complex in the DNA damage response in Aspergillus nidulans , 2003, Molecular microbiology.

[53]  Ari Barzilai,et al.  DNA damage responses to oxidative stress. , 2004, DNA repair.

[54]  G. Goldman,et al.  The Aspergillus nidulans npkA Gene Encodes a Cdc2-Related Kinase That Genetically Interacts With the UvsBATR Kinase Sequence data from this article have been deposited with the NCBI under accession no. AY166593. , 2004, Genetics.

[55]  L. Travassos,et al.  Identification of genes preferentially expressed in the pathogenic yeast phase of Paracoccidioides brasiliensis, using suppression subtraction hybridization and differential macroarray analysis , 2004, Molecular Genetics and Genomics.

[56]  P. Koprowski,et al.  Repair of oxidative damage in mitochondrial DNA of Saccharomyces cerevisiae: involvement of the MSH1-dependent pathway. , 2004, DNA repair.

[57]  P. Mirabito,et al.  The early impact of genetics on our understanding of cell cycle regulation in Aspergillus nidulans. , 2004, Fungal genetics and biology : FG & B.

[58]  G. Goldman,et al.  Aspergillus nidulans as a model system to characterize the DNA damage response in eukaryotes. , 2004, Fungal genetics and biology : FG & B.

[59]  G. de Murcia,et al.  The PARP superfamily , 2004, BioEssays : news and reviews in molecular, cellular and developmental biology.

[60]  S. Jazwinski.  Yeast replicative life span--the mitochondrial connection. , 2004, FEMS yeast research.

[61]  J. Hoeijmakers,et al.  Different Effects of CSA and CSB Deficiency on Sensitivity to Oxidative DNA Damage , 2004, Molecular and Cellular Biology.

[62]  M. Y. Kim,et al.  Poly(ADP-ribosyl)ation by PARP-1: 'PAR-laying' NAD+ into a nuclear signal. , 2005, Genes & development.

[63]  D. Loegering,et al.  The Role of Checkpoint Kinase 1 in Sensitivity to Topoisomerase I Poisons* , 2005, Journal of Biological Chemistry.

[64]  C. P. Semighini,et al.  Aspergillus nidulans uvsB and scaA Genes Show Genetic Interactions during Recovery from Replication Stress and DNA Damage , 2005 .

[65]  M. Z. Humayun,et al.  The Helicobacter pylori MutS protein confers protection from oxidative DNA damage , 2005, Molecular microbiology.

[66]  Christina A. Cuomo,et al.  Sequencing of Aspergillus nidulans and comparative analysis with A. fumigatus and A. oryzae , 2005, Nature.

[67]  C. P. Semighini,et al.  Aspergillus nidulans uvsBATR and scaANBS1 Genes Show Genetic Interactions during Recovery from Replication Stress and DNA Damage , 2005, Eukaryotic Cell.

[68]  N. Nowak,et al.  In vitro and in vivo irinotecan-induced changes in expression profiles of cell cycle and apoptosis-associated genes in acute myeloid leukemia cells , 2005, Molecular Cancer Therapeutics.

[69]  Eyal Gottlieb,et al.  Mitochondrial tumour suppressors: a genetic and biochemical update , 2005, Nature Reviews Cancer.

[70]  Csaba Szabó,et al.  Poly(ADP-ribose) polymerase and the therapeutic effects of its inhibitors , 2005, Nature Reviews Drug Discovery.

[71]  L. Murphy,et al.  Prohibitin: a potential target for new therapeutics. , 2005, Trends in molecular medicine.

[72]  C. P. Semighini,et al.  Regulation of Hyphal Morphogenesis and the DNA Damage Response by the Aspergillus nidulans ATM Homolog AtmA , 2006, Genetics.

[73]  R. Serra,et al.  A cDNA-microarray analysis of camptothecin resistance in glioblastoma cell lines. , 2006, Cancer letters.

[74]  Xu-ping Fu,et al.  Analysis of common gene expression patterns in four human tumor cell lines exposed to camptothecin using cDNA microarray: identification of topoisomerase-mediated DNA damage response pathways. , 2006, Frontiers in bioscience : a journal and virtual library.

[75]  S. Osmani,et al.  A Versatile and Efficient Gene-Targeting System for Aspergillus nidulans , 2006, Genetics.

[76]  C. P. Semighini,et al.  Functional Characterization of the Putative Aspergillus nidulans Poly(ADP-Ribose) Polymerase Homolog PrpA , 2006, Genetics.