Mutational analysis of Brh2 reveals requirements for compensating mediator functions - PubMed (original) (raw)

Mutational analysis of Brh2 reveals requirements for compensating mediator functions

Milorad Kojic et al. Mol Microbiol. 2011 Jan.

Abstract

Brh2, a member of the BRCA2 family of proteins, governs homologous recombination in the fungus Ustilago maydis through interaction with Rad51. Brh2 serves at an early step in homologous recombination to mediate Rad51 nucleoprotein filament formation and also has the capability to function at a later step in recombination through its inherent DNA annealing activity. Rec2, a Rad51 paralogue, and Rad52 are additional components of the homologous recombination system, but the absence of either is less critical than Brh2 for operational activity. Here we tested a variety of mutant forms of Brh2 for activity in recombinational repair as measured by DNA repair proficiency. We found that a mutant of Brh2 deleted of the non-canonical DNA-binding domain within the N-terminal region is dependent upon the presence of Rad52 for DNA repair activity. We also determined that a motif first identified in human BRCA2 as important in binding DMC1 also contributes to DNA repair proficiency and cooperates with the BRC element in Rad51 binding.

© 2010 Blackwell Publishing Ltd.

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Figures

Figure 1

Figure 1

Transposon scanning mutagenesis. A. Schematic representation of Brh2 with protein elements and domains as follows: BRC -- Rad51 interaction element; NBD -- N-terminal DNA binding domain; HD -- helix rich domain; OB1--OB module; OB2/tower- OB module with tower insert; CRE – Rad51 interaction region. Sites of 15 bp insertions, with corresponding translations, introduced after excision of the indicated transposons are shown. B. Mutant strains expressing the indicated Brh2 were grown to late log phase. Aliquots were adjusted to a density of 2 × 107 cells per ml, spotted on solid medium as serial 10-fold dilutions from left to right, and survival was determined after irradiation with UV at a dose of 120 J/m2.

Figure 2

Figure 2

Brh2 deletion mapping and domain swapping. A. Schematic representations of Brh2’s with various fragments deleted or replaced by DNA binding modules OB-A and OB-B from U. maydis RPA70. B. Mutant strains expressing the indicated Brh2 constructs were tested for survival after irradiation with UV at a dose of 120 J/m2.

Figure 3

Figure 3

Contribution of Phe(P/T)P and FTTP motifs to DNA repair activity. A. Schematic representation of Brh2 with mutated residues in BRC, Phe(P/T)P, and FTTP sequence motifs. brh2 strains expressing the indicated Brh2 point mutation constructs were tested for survival after irradiation with UV at a dose of 120 J/m2. B. Schematic representation of Brh2ΔC551 with Phe(P/T)P point mutations. brh2 expressing the indicated Brh2ΔC551 constructs were tested for survival. C. Schematic representation of Brh2 with Phe(P/T)P and CRE point mutations. Constructs were tested for survival.

Figure 4

Figure 4

Mutation in Brh2 Phe(P/T)P motif disturbs Rad51 binding. A. Schematic representation shows Brh2ΔC551 with mutated residues in BRC and Phe(P/T)P. Brh2ΔC551 and derivatives were tested for ability to form complexes with Rad51. Pulldowns of MBP-tagged Brh2ΔC551 variants plus associated Rad51 protein were performed as described in the experimental procedures. Left panel- preload samples; right panel-complexes eluted from amylose resin with maltose. Relative levels of Rad51 and Brh2ΔC551 were quantified by comparing the band intensity after staining. Averages of triplicates are presented with the standard error. Variants of Brh2ΔC551 with indicated mutations are as follows: lane a-wild type (wt); b-F294A T296A; c-F488A; d- F294A T296A F488A; e-no Brh2ΔC551 variant. B. Schematic representation shows Brh2ΔN359 with mutated residues F488A in Phe(P/T)P and W1052 in CRE. Pulldowns of MBP-tagged Brh2ΔN359 variants plus associated Rad51 protein from cleared E. coli extracts were performed as in experimental procedures. Left panel- preload samples; right panel-complexes eluted from amylose resin with maltose. Relative levels of Rad51 and Brh2ΔC551 were quantified by comparing the band intensity after ECL development. lane a-wild type (wt); b-F488A; c-W1052A; d-F488A W1052A; e-no Brh2ΔN359 variant.

Figure 5

Figure 5

DNA repair proficiency of Brh2 Phe(P/T)P and FTTP point mutations in the absence of Rec2 function. A. rec2 strains expressing the indicated Brh2 point mutation constructs were tested for survival after irradiation with UV at a dose of 120 J/m2. B Schematic representation of Rec2 (781 amino acids) RecA-related sequence and lysine residue K257 within the Walker A motif. rec2 expressing the indicated Rec2 K257R or K257A mutant constructs or co-expressing the Brh2 F488A Phe(P/T)P motif mutant and Rec2 point mutants were tested for survival. C. rec2 strains expressing Brh2ΔC551 with the indicated mutations were tested for survival after irradiation with gamma rays at a dose of 400 Gy.

Figure 6

Figure 6

Contribution of Rad52 domains to DNA repair proficiency of Bhr2 deleted of NBD. A. Schematic showing Brh2 deleted of NBD and Rad52 with its DNA binding domain (DBD) plus N- and C-terminal truncations. B. Survival of brh2 rad52 mutant strain expressing the indicated constructs after irradiation with UV at a dose of 120 J/m2.

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