In a screen for DNA repair-defective mutants in the fungus Ustilago maydis, a gene encoding a BRCA2 family member, designated here as Brh2, was identified. A brh2 null allele was found to be defective in allelic recombination, meiosis, and repair of gaps and ionizing radiation damage to the same extent as rad51. Frequent marker loss in meiosis and diploid formation suggested that genomic instability was associated with brh2. This notion was confirmed by molecular karyotype analysis, which revealed gross chromosomal alterations associated with brh2. Yeast two-hybrid analysis indicated interaction between Brh2 and Rad51. Recapitulation in U. maydis of defects in DNA repair and genome stability associated with brh2 means that the BRCA2 gene family is more widespread than previously thought.

DSS1 encodes a small acidic protein shown in recent structural studies to interact with the DNA binding domain of BRCA2. Here we report that an ortholog of DSS1 is present in Ustilago maydis and associates with Brh2, the BRCA2-related protein, thus recapitulating the protein partnership in this genetically amenable fungus. Mutants of U. maydis deleted of DSS1 are extremely radiation sensitive, deficient in recombination, defective in meiosis, and disturbed in genome stability; these phenotypes mirror previous observations of U. maydis mutants deficient in Brh2 or Rad51. These findings conclusively show that Dss1 constitutes a protein with a significant role in the recombinational repair pathway in U. maydis, and imply that it plays a similar key role in the recombination systems of organisms in which recombinational repair is BRCA2 dependent.

Brh2, the BRCA2 homolog in Ustilago maydis, functions in recombinational repair of DNA damage by regulating Rad51 and is, in turn, regulated by Dss1. Dss1 is not required for Brh2 stability in vivo, nor for Brh2 to associate with Rad51, but is required for formation of green fluorescent protein (GFP)-Rad51 foci following DNA damage by gamma radiation. To understand more about the interplay between Brh2 and Dss1, we isolated mutant variants of Brh2 able to bypass the requirement for Dss1. These variants were found to lack the entire C-terminal DNA-Dss1 binding domain but to maintain the N-terminal region harboring the Rad51-interacting BRC element. GFP-Rad51 focus formation was nearly normal in brh2 mutant cells expressing a representative Brh2 variant with the C-terminal domain deleted. These findings suggest that the N-terminal region of Brh2 has an innate ability to organize Rad51. Survival after DNA damage was almost fully restored by a chimeric form of Brh2 having a DNA-binding domain from RPA70 fused to the Brh2 N-terminal domain, but Rad51 focus formation and mitotic recombination were elevated above wild-type levels. The results provide evidence for a mechanism in which Dss1 activates a Brh2-Rad51 complex and balances a finely regulated recombinational repair system. BRCA2 is the product of a major breast cancer susceptibility gene in humans functioning in homology-directed repair of DNA through association with the Rad51 recombinase that provides the essential activity required for DNA strand exchange. In vertebrate systems, regulated interaction between Rad51 and BRCA2 is critical for repair of DNA double-strand breaks (31). There is support for two primary levels of control. First, evidence has accumulated pointing to a role for BRCA2 in nuclear localization of Rad51 (10,24,30). Second, assembly of Rad51 into its catalytically active form, the nucleoprotein filament generated through Rad51 polymerization on singlestranded DNA, is regulated by BRCA2 (10,30,32,43,44). Filament assembly is balanced on the one hand by the inherent ability of Rad51 monomers to self-associate coupled with BRCA2's activity initiating the association and on the other hand by attenuation of assembly and destabilization of the filament through interference with a crucial contact between Rad51 subunits via BRCA2's BRC elements (32,36,43). The latter are a series of eight related sequences of about 30 residues each that mimic a Rad51 motif at the polymerization interface. Studies on the dynamics of green fluorescent protein (GFP)-tagged protein trafficking in the nuclei of living cells after photobleaching suggest that Rad51 is sequestered by BRCA2 in an immobilized form awaiting activation by DNA damage (44). The emerging model features BRCA2 as a governor of Rad51 nuclear localization and Rad51-mediated homology-directed repair (31), although evidence linking BRCA2 deficiency to abnormal centrosome formation (24,40) and cytokinesis (9) suggests that this view may be oversimplified.BRCA2 has a DNA-binding domain (DBD) contained w...