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 (ncRAD5 2) by PCR, using degenerate primers. RFLP mapping demonstrated that the clo ned gene is located close to the ro-4 locus on the fight arm of linkage gro up V (LGVR). In a second experiment, we used sib selection to identify a co smid 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 nuc leotide 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 sequen ce of the MUS11 protein shows 32.0% and 27.5% overall identity to the Schiz osaccharomyces pombe Rad22 protein and the human hRad52 protein, respective ly, and a higher level of identity (55-66%) within the conserved N-terminal region (141 residues). The MUS11 protein shows homology to Rad52 from budd ing 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 ho mologue, and to itself in vivo. An ncRAD52 mutant obtained by the RIPping p rocedure showed the same sensitivity as the original mus-11 mutant to the f ollowing mutagens and chemicals: UV light, 4NQO (4-nitroquinoline 1-oxide), MMS (methyl methanesulfonate), EMS (ethyl methanesulfonate), MNNG (N-methy l-N'-nitro-N-nitrosoguanidine), TBHP (tert-butyl hydroperoxide), HU (hydrox yurea) and histidine. Unlike the RAD52 transcript in Saccharomyces cerevisi ae, the mus-11 transcript could not be detected in mycelium under normal gr owth conditions, but expression of the gene was induced by UV irradiation o r treatment with MMS.