Sensitivity to camptothecin in Aspergillus nidulans identifies a novel gene, scaA + , related to the cellular DNA damage
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O R I GI N A L P A P E R G. C. M. Bruschi á C. C. de Souza M. R. von Z. K. Fagundes á M. A. C. Dani M. H. S. Goldman á N. R. Morris L. Liu á G. H. Goldman
Sensitivity to camptothecin in Aspergillus nidulans identi®es a novel gene, scaA +, related to the cellular DNA damage response Received: 21 August 2000 / Accepted: 25 October 2000 / Published online: 19 January 2001 Ó Springer-Verlag 2001
Abstract 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 ®lamentous fungus Aspergillus nidulans and characterised one of them, sca299. The mutant sca299 is hypersensitive to camptothecin, and sensitive to several dierent 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 aected 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
G. C. M. Bruschi á C. C. de Souza M. R. von Z. K. Fagundes á M. A. C. Dani G. H. Goldman (&) Faculdade de CieÃncias FarmaceÃuticas de RibeiraÄo Preto, Universidade de SaÄo Paulo, Av. do Cafe S/N, CEP 14040-903, RibeiraÄo Preto, SaÄo Paulo, Brazil E-mail: [email protected] Tel.: +55-016-6024280/81 Fax: +55-016-6331092 M. H. S. Goldman Faculdade de Filoso®a, CieÃncias e Letras de RibeiraÄo Preto, Universidade de SaÄo Paulo, Brazil N. R. Morris á L. Liu Department of Pharmacology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, NJ 08854, USA
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. Key words Camptothecin á Aspergillus nidulans á DNA damage á DNA repair
Introduction Topoisomerases are enzymes that modify and regulate the topological state of DNA. They are required for replication, transcription and recombination, and play critical roles in chromosome structure, condensation/ decondensation, and segregation (Liu 1989; Berger 1998). Beyond the
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