Dss1 Interaction with Brh2 as a Regulatory Mechanism for Recombinational Repair

Abstract

Brh2, the BRCA2 ortholog in Ustilago maydis, enables recombinational repair of DNA by controlling Rad51 and is in turn regulated by Dss1. Interplay with Rad51 is conducted via the BRC element located in the N-terminal region of the protein and through an unrelated domain, CRE, at the C terminus. Mutation in either BRC or CRE severely reduces functional activity, but repair deficiency of the brh2 mutant can be complemented by expressing BRC and CRE on different molecules. This intermolecular complementation is dependent upon the presence of Dss1. Brh2 molecules associate through the region overlapping with the Dss1-interacting domain to form at least dimer-sized complexes, which in turn, can be dissociated by Dss1 to monomer. We propose that cooperation between BRC and CRE domains and the Dss1-provoked dissociation of Brh2 complexes are requisite features of Brh2's molecular mechanism.Accumulating evidence points to a choreographed interaction between Rad51 and BRCA2 as a critical mechanism governing recombinational repair (9,21,27,30). Proper regulation of the repair process requires assembly of Rad51 into its catalytically active form, the nucleoprotein filament. This filament is generated through Rad51's polymerization on singlestranded DNA, a process that appears to be mediated by BRCA2. Biochemical analyses using Brh2, the BRCA2-related protein from Ustilago maydis, have demonstrated that it nucleates Rad51 assembly at the site of a double-strand/singlestrand DNA junction, the prerequisite structure for recombinational repair arising from resection of a double-strand DNA end to reveal a protruding 3Ј single-stranded tail (32). However, molecular genetic experimentation with U. maydis (15) as well as biochemical studies using peptides modeling BRC elements from the human BRCA2 (6) or a polypeptide containing multiple BRC elements (26) suggest a role for BRCA2 in organization or stabilization of Rad51 filaments.Regulated assembly of the filament may be balanced on the one hand by interaction of Rad51 with BRC elements and on the other hand by some interplay with a second domain located at the extreme C terminus of BRCA2 (CRE [C-terminal Rad51-interacting element]; see below) (4). The BRC domain comprises eight reiterated sequences of about 30 amino acids each whose structures have been proposed to mimic an element in Rad51 that provides a critical determinant at the polymerization interface, gluing Rad51 molecules into a chain (20,25). Rad51 interaction with the C-terminal domain appears to be controlled by phosphorylation of a key BRCA2 residue that is targeted by cyclin-dependent kinases (4). Proper Rad51 filament assembly at DNA sites of repair requires a precisely coordinated interplay between BRCA2's Rad51-interacting domains and its DSS1/DNA-binding domain (DBD). The latter (31) consists of a tandem array of OB (oligonucleotide/oligosaccharide-binding) folds, with a double-helical tower emerging from one OB fold topped by a helix-turn-helix, and a helical domain that is laced to the adjacent OB folds by ...

Ortholog of BRCA2-interacting protein BCCIP controls morphogenetic responses during DNA replication stress in Ustilago maydis

“…However, dss1 and bcp1 mutants are both defective in cell growth ability, although dss1 is not as strongly disturbed as bcp1 (compare this work with [22]). Similar to the previously reported observation that the dss1 brh2 double mutant grows better than the dss1 single mutant [31], here we also noted that the bcp1 brh2 double mutant grew more robustly than the bcp1 single mutant. In the case of dss1 we interpreted the observations to mean that Brh2 confers toxicity in the absence of Dss1, perhaps by interfering with the normal DNA processing systems [31].…”

“…Similar to the previously reported observation that the dss1 brh2 double mutant grows better than the dss1 single mutant [31], here we also noted that the bcp1 brh2 double mutant grew more robustly than the bcp1 single mutant. In the case of dss1 we interpreted the observations to mean that Brh2 confers toxicity in the absence of Dss1, perhaps by interfering with the normal DNA processing systems [31]. Since Dss1 has been reported to play a role as a nuclear transporter [36], the notion occurred that one means of regulating Brh2 and mitigating this toxicity might be for Dss1 to shuttle Brh2 out of the nucleus.…”

“…Expression of MBP-Brh2 and His-Bcp1 fusion proteins in E. coli strain BL21(DE3), and coprecipitation or pull-down analyses have been described previously [26,31]. As deletions removing up to 213 amino acid residues from the N-terminus of Brh2 make no difference in ability to complement the radiation sensitivity of the brh2 null mutant [32], we cut into the coding sequence for this nonessential region to expedite cloning of Brh2 truncation alleles and to enhance solubility of their protein products.…”

DNA-binding Domain within the Brh2 N Terminus Is the Primary Interaction Site for Association with DNA

“…maydis Genetic Methods-Manipulations of U. maydis strains, culture methods, gene transfer procedures, and survival after UV and ␥ radiation have been described previously (13,21). U. maydis strains included UCM350 (wild type for recombination function), UCM565 (brh2), and UCM54 (rec2).…”

“…The BRC element comprises a sequence of about 30 amino acids (aa) that mimics a critical determinant of the Rad51 polymerization interface (11,12). Rad51 also interacts with Brh2 through a second, poorly defined locus, the CRE, located at the extreme C terminus of Brh2 (13). These N-terminal and C-terminal elements communicate with each other in some as yet unknown way to support proper Rad51 filament assembly.…”