Highly efficient gene replacements in Neurospora strains deficient for nonhomologous end-joining.

Gene disruption and overexpression play central roles in the analysis of gene function. Homologous recombination is, in principle, the most efficient method of disrupting, modifying, or replacing a target gene. Although homologous integration of exogenous DNA into the genome occurs readily in Saccharomyces cerevisiae, it is rare in many other organisms. We identified and disrupted Neurospora crassa genes homologous to human KU70 and KU80, which encode proteins that function in nonhomologous end-joining of double-stranded DNA breaks. The resulting mutants, named mus-51 and mus-52, showed higher sensitivity to methyl methanesulfonate, ethyl methanesulfonate, and bleomycin than wild type, but not to UV, 4-nitroquinoline 1-oxide, camptothecin, or hydroxyurea. Vegetative growth, conidiation, and ascospore production in homozygous crosses were normal. The frequency of integration of exogenous DNA into homologous sequences of the genome in the KU disruption strains of N. crassa was compared with that in wild type, mei-3, and mus-11. In mei-3 and mus-11, which are defective in homologous recombination, none or few homologous integration events were observed under any conditions. When mtr target DNA with approximately 2-kb 5' and 3' flanking regions was used for transformation of the KU disruption strains, 100% of transformants exhibited integration at the homologous site, compared to 10 to 30% for a wild-type recipient. Similar results were obtained when the ad-3A gene was targeted for disruption. These results indicate that KU disruption strains are efficient recipients for gene targeting.

[1]  E. Mauceli,et al.  The genome sequence of the filamentous fungus Neurospora crassa , 2003, Nature.

[2]  J. Wendland.  PCR-based methods facilitate targeted gene manipulations and cloning procedures , 2003, Current Genetics.

[3]  H. Inoue,et al.  The Neurospora crassa mus-19 gene is identical to the qde-3 gene, which encodes a RecQ homologue and is involved in recombination repair and postreplication repair , 2004, Current Genetics.

[4]  H. Inoue,et al.  Isolation and characterization of a laccase-derepressed mutant of Neurospora crassa , 1989, Journal of bacteriology.

[5]  S. Tanksley,et al.  Abundance, polymorphism and genetic mapping of microsatellites in rice , 1993, Molecular and General Genetics MGG.

[6]  T. Petes,et al.  Effect of mutations in genes affecting homologous recombination on restriction enzyme-mediated and illegitimate recombination in Saccharomyces cerevisiae , 1994, Molecular and cellular biology.

[7]  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.

[8]  Rowland H. Davis,et al.  [4] Genetic and microbiological research techniques for Neurospora crassa , 1970 .

[9]  H. Sambrook.  Molecular cloning : a laboratory manual. Cold Spring Harbor, NY , 1989 .

[10]  Michael Freitag,et al.  Lessons from the Genome Sequence of Neurospora crassa: Tracing the Path from Genomic Blueprint to Multicellular Organism , 2004, Microbiology and Molecular Biology Reviews.

[11]  M. Orbach,et al.  A cosmid with a HyR marker for fungal library construction and screening. , 1994, Gene.

[12]  H. Inoue,et al.  Identification and expression of the Neurospora crassa mei-3 gene which encodes a protein homologous to Rad51 of Saccharomyces cerevisiae , 1995, Molecular and General Genetics MGG.

[13]  G. Macino,et al.  Characterization of the Neurospora crassa mus-25 mutant: the gene encodes a protein which is homologous to the Saccharomyces cerevisiae Rad54 protein , 2000, Molecular and General Genetics MGG.

[14]  W. Dynan,et al.  Interaction of Ku protein and DNA-dependent protein kinase catalytic subunit with nucleic acids. , 1998, Nucleic acids research.

[15]  P. Philippsen,et al.  New heterologous modules for classical or PCR‐based gene disruptions in Saccharomyces cerevisiae , 1994, Yeast.

[16]  T. Mimori,et al.  Characterization of a high molecular weight acidic nuclear protein recognized by autoantibodies in sera from patients with polymyositis-scleroderma overlap. , 1981, The Journal of clinical investigation.

[17]  H. Inoue,et al.  Genetic and molecular characterization of Neurospora crassamus-23 :a gene involved in recombinational repair , 1997, Molecular and General Genetics MGG.

[18]  H. Inoue,et al.  Isolation and characterization of MMS-sensitive mutants of Neurosporacrassa , 1984 .

[19]  K. Tamura,et al.  Identification of Ku70 and Ku80 homologues in Arabidopsis thaliana: evidence for a role in the repair of DNA double-strand breaks. , 2002, The Plant journal : for cell and molecular biology.

[20]  Y. Anraku,et al.  Applications of the Long and Accurate Polymerase Chain Reaction Method in Yeast Molecular Biology: Direct Sequencing of the Amplified DNA and Its Introduction into Yeast , 1997, Yeast.

[21]  E. Selker.  Premeiotic instability of repeated sequences in Neurospora crassa. , 1990, Annual review of genetics.

[22]  J. Walker,et al.  Structure of the Ku heterodimer bound to DNA and its implications for double-strand break repair , 2001, Nature.

[23]  S. Jackson,et al.  DNA end-joining: from yeast to man. , 1998, Trends in biochemical sciences.

[24]  C. Yanofsky,et al.  Efficient cloning of genes of Neurospora crassa. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[25]  H. Inoue,et al.  The Neurospora uvs-2 gene encodes a protein which has homology to yeast RAD18, with unique zinc finger motifs , 1993, Molecular and General Genetics MGG.