A Neurospora double-strand-break repair gene, mus-11, encodes a RAD52 homologue and is inducible by mutagens.
We isolated a Neurospora crassa cDNA that encodes a Rad52 homologue (ncRAD52) by PCR, using degenerate primers. RFLP mapping demonstrated that the cloned gene is located close to the ro-4 locus on the right arm of linkage group V (LGVR). In a second experiment, we used sib selection to identify a cosmid clone containing the mus-11 gene in a N. crassa genomic library. Fine-scale mapping of the mus-11 mutant showed the gene order on LGVR near ro-4 to be: ad-7 - (9.5 mu) - pab-2 (7.8 mu) - mus-11 - (3.7 mu) - inv. The nucleotide sequence of the mus-11 gene matched that of the ncRAD52 cDNA. Thus, the mus-11 gene encodes the Rad52 homologue. The deduced amino acid sequence of the MUS11 protein shows 32.0% and 27.5% overall identity to the Schizosaccharomyces pombe Rad22 protein and the human hRad52 protein, respectively, and a higher level of identity (55-66%) within the conserved N-terminal region (141 residues). The MUS11 protein shows homology to Rad52 from budding yeast only within the N-terminal region (53.2% identity over 141 amino acids) which is conserved among Rad52 homologues. Yeast two-hybrid analysis reveals that the MUS11 protein binds to both the MEI-3 protein, a Rad51 homologue, and to itself in vivo. An ncRAD52 mutant obtained by the RIPping procedure showed the same sensitivity as the original mus-11 mutant to the following mutagens and chemicals: UV light, 4NQO (4-nitroquinoline 1-oxide), MMS (methyl methanesulfonate), EMS (ethyl methanesulfonate), MNNG (N-methyl-N'-nitro-N-nitrosoguanidine), TBHP (tert-butyl hydroperoxide), HU (hydroxyurea) and histidine. Unlike the RAD52 transcript in Saccharomyces cerevisiae, the mus-11 transcript could not be detected in mycelium under normal growth conditions, but expression of the gene was induced by UV irradiation or treatment with MMS.
Sakuraba
Y
Y
Department of Regulation-Biology, Faculty of Science, Saitama University, Urawa, Japan.
Schroeder
A L
AL
Ishii
C
C
Inoue
H
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004260:DNA Repair / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification; D004268:DNA-Binding Proteins; D004706:Endodeoxyribonucleases; D004843:Epistasis, Genetic; D005656:Fungal Proteins / Q000235:genetics* / Q000502:physiology*; D005800:Genes, Fungal / Q000187:drug effects / Q000528:radiation effects; D006801:Humans; D008741:Methyl Methanesulfonate / Q000633:toxicity; D008969:Molecular Sequence Data; D009153:Mutagens / Q000633:toxicity; D009492:Neurospora crassa / Q000187:drug effects / Q000235:genetics* / Q000528:radiation effects; D012150:Polymorphism, Restriction Fragment Length; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D017386:Sequence Homology, Amino Acid; D014466:Ultraviolet Rays
D017931:DNA Primers; D018076:DNA, Complementary; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C116305:MEI3 protein, S pombe; C417728:MUS11 protein, Neurospora crassa; D009153:Mutagens; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; C073998:RAD2 protein, S cerevisiae; D008741:Methyl Methanesulfonate; D004706:Endodeoxyribonucleases