Rec2 Interplay with both Brh2 and Rad51 Balances Recombinational Repair in Ustilago maydis

Abstract

Rec2 is the single Rad51 paralog in Ustilago maydis. Here, we find that Rec2 is required for radiation-induced Rad51 nuclear focus formation but that Rec2 foci form independently of Rad51 and Brh2. Brh2 foci also form in the absence of Rad51 and Rec2. By coprecipitation from cleared extracts prepared from Escherichia coli cells expressing the proteins, we found that Rec2 interacts physically not only with Rad51 and itself but also with Brh2. Transgenic expression of Brh2 in rec2 mutants can effectively restore radiation resistance, but the frequencies of spontaneous Rad51 focus formation and allelic recombination are elevated. The Dss1-independent Brh2-RPA70 fusion protein is also active in restoring radiation sensitivity of rec2 but is hyperactive to an extreme degree in allelic recombination and in suppressing the meiotic block of rec2. However, the high frequency of chromosome missegregation in meiotic products is an indicator of a corrupted process. The results demonstrate that the importance of Rec2 function is not only in stimulating recombination activity but also in ensuring that recombination is properly controlled.Repair of DNA damaged by double-strand breakage or replication fork collapse can take advantage of a homologous sequence for use as a template in directing accurate correction. Rad51 provides the essential homologous pairing and DNA strand exchange activity required for homology-directed or recombinational repair (53). In mitotic cells, Rad51 alone is sufficient to power homologous pairing (47), while in meiotic cells, Dmc1 can contribute through its own innate homologous-pairing activity (6, 43). Accumulating evidence points to a choreographed interaction between Rad51 and BRCA2 as a critical mechanism governing recombinational repair (25,40,48). Assembly of Rad51 into its catalytically active form, the nucleoprotein filament generated through Rad51 polymerization on single-stranded DNA, appears to be regulated both positively and negatively by BRCA2. There is evidence for control at three levels of Rad51 filament dynamics. Biochemical analyses using Brh2, the BRCA2-related protein from Ustilago maydis, demonstrate that it can function to nucleate Rad51 assembly at the site of a double-strand/single-strand 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 (60). On the other hand, molecular genetic experimentation using U. maydis (31) as well as biochemical studies using synthetic peptides modeling BRC elements from the human BRCA2 (21) suggest a role in organizing or stabilizing Rad51 filaments. In contrast, work with BRC peptides has also provided evidence for a role in filament disassembly (14), interference with Rad51 focus formation (11), and inhibition of recombination and repair (51), although these effects could have resulted from the very high levels of BRC peptides used.Regulated assembly of the filament appears balanced on the one hand by the interaction of Rad51 with ...

Dss1 Interaction with Brh2 as a Regulatory Mechanism for Recombinational Repair

“…Plasmids with a variety of antibiotic resistance markers used for gene disruption and protein expression in U. maydis have been described previously (11,16). These are pUC derivatives containing an U. maydis ARS gene, a fragment of the glyceraldehyde-3-phosphate dehydrogenase promoter (gap) used for driving expression of Brh2 alleles, and a gene expressing resistance to hygromycin (Hyg R ), gentamicin (G418 R ), or carboxin (Cbx R ) for use as a selectable marker.…”

“…Protein expression, coprecipitation, purification from Escherichia coli, and cross-linking. Pull-down analyses were performed using bacterial extracts prepared after expression of maltose binding protein (MBP)-tagged or His-tagged fusion proteins in E. coli strain BL21(DE3) (16). As deletions removing up to 213 amino acid residues from the N terminus of Brh2 make no difference in the ability to complement the radiation sensitivity of the brh2 null mutant (15), we cut into the coding sequence for this nonessential region to expedite cloning of Brh2 truncation alleles and to enhance the solubility of their protein products.…”

“…By using a coprecipitation (pulldown) procedure to map Rad51-interacting regions in Brh2, we demonstrated the capability of the N-terminal BRC to bind Rad51 and confirmed the importance of certain canonical residues in the interaction (16). As our previous deletion mapping of Brh2 demonstrated that the removal of 41 residues from the C terminus was enough to abolish the activity of Brh2 in promoting survival after DNA damage (15), we were curious to know if this deletion disrupted a Rad51-binding domain or element (CRE) analogous to that found at the extreme C terminus of BRCA2 and, if so, whether it might be responsible in part for the putative negative regulatory function.…”

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

“…Manipulations with U. maydis, culture methods, diploid construction, gene transfer procedures, survival after irradiation, and recombination assays have been described previously (see [26] and references therein). The gene encoding Bcp1 was identified as um03018 in the annotated Ustilago maydis genome database (http://mips.gsf.de/genre/proj/ustilago/) (GenBank accession number EF382649).…”

“…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.…”