The Neurospora crassa UVS-3 epistasis group encodes homologues of the ATR/ATRIP checkpoint control system.
The mutagen sensitive uvs-3 and mus-9 mutants of Neurospora show mutagen and hydroxyurea sensitivity, mutator effects and duplication instability typical of recombination repair and DNA damage checkpoint defective mutants. To determine the nature of these genes we used cosmids from a genomic library to clone the uvs-3 gene by complementation for MMS sensitivity. Mutation induction by transposon insertion and RIP defined the coding sequence. RFLP analysis confirmed that this sequence maps in the area of uvs-3 at the left telomere of LG IV. Analysis of the cDNA showed that the UVS-3 protein contains an ORF of 969 amino acids with one intron. It is homologous to UvsD of Aspergillus nidulans, a member of the ATRIP family of checkpoint proteins. It retains the N' terminal coiled-coil motif followed by four basic amino acids typical of these proteins and shows the highest homology in this region. The uvsD cDNA partially complements the defects of the uvs-3 mutation. The uvs-3 mutant shows a higher level of micronuclei in conidia and failure to halt germination and nuclear division in the presence of hydroxyurea than wild type, suggesting checkpoint defects. ATRIP proteins bind tightly to ATR PI-3 kinase (phosphatidylinositol 3-kinase) proteins. Therefore, we searched the Neurospora genome sequence for homologues of the Aspergillus nidulans ATR, UvsB. A uvsB homologous sequence was present in the right arm of chromosome I where the mus-9 gene maps. A cosmid containing this genomic DNA complemented the mus-9 mutation. The putative MUS-9 protein is 2484 amino acids long with eight introns. Homology is especially high in the C-terminal 350 amino acids that correspond to the PI-3 kinase domain. In wild type a low level of constitutive mRNA is present for both genes. It is transiently induced upon UV exposure.
Kazama
Yusuke
Y
Laboratory of Genetics, Department of Regulation Biology, Faculty of Sciences, Saitama University, Saitama, Japan.
Ishii
Chizu
C
Schroeder
Alice L
AL
Shimada
Hisao
H
Wakabayashi
Michiyoshi
M
Inoue
Hirokazu
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D018797:Cell Cycle Proteins / Q000235:genetics*; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000235:genetics*; D004843:Epistasis, Genetic; D005656:Fungal Proteins / Q000235:genetics*; D007700:Kinetics; D008741:Methyl Methanesulfonate; D008969:Molecular Sequence Data; D009492:Neurospora crassa / Q000235:genetics*; D016366:Open Reading Frames / Q000235:genetics; D017354:Point Mutation / Q000235:genetics; D012150:Polymorphism, Restriction Fragment Length; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D014466:Ultraviolet Rays
D018797:Cell Cycle Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D008741:Methyl Methanesulfonate