Analysis of the indispensable RAD51 cofactor BRCA2 in Naganishia liquefaciens, a Basidiomycota yeast

Abstract

The BRCA2 tumor suppressor plays a critical role in homologous recombination by regulating RAD51, the eukaryotic homologous recombinase. We identified the BRCA2 homolog in a Basidiomycota yeast, Naganishia liquefaciens BRCA2 homologs are found in many Basidiomycota species but not in Ascomycota species. Naganishia BRCA2 (Brh2, for BRCA2 homolog) is about one-third the size of human BRCA2. Brh2 carries three potential BRC repeats with two oligonucleotide/oligosaccharide-binding domains. The homolog of DSS1, a small acidic protein serving as an essential partner of BRCA2 was also identified. The yeast two-hybrid assay shows the interaction of Brh2 with both Rad51 and Dss1. Unlike human BRCA2, Brh2 is not required for normal cell growth, whereas loss of Dss1 results in slow growth. The loss of Brh2 caused pronounced sensitivity to UV and ionizing radiation, and their HR ability, as assayed by gene-targeting efficiency, is compromised. These phenotypes are indistinguishable from those of the rad51 mutant, and the rad51 brh2 double mutant. Naganishia Brh2 is likely the BRCA2 ortholog that functions as an indispensable auxiliary factor for Rad51.

Figure 1.. BRCA2 homologs in N. liquefaciens and other Basidiomycota yeasts.

(A) Schematic representation of H. sapiens (P51587) BRCA2 and its homologs in N. liquefaciens (GHJ86783), C. neoformans (UOH82896), and U. maydis (XP_011389639). (B) Alignment of potential BRC repeats from three Basidiomycota yeasts. Nl, N. liquefaciens; Cn, C. neoformans; Um, U. maydis. Numbers assigned to BRC repeats correspond to the numbering in (A). Φ, any hydrophobic amino acid. (C) Taxonomical distribution of BRCA2 homologs. Refer to Table S1 for more details. (D) Structural models of the BRCA2 C-terminal region of human (Hs) and N. liquefaciens (Nl) generated by AlphaFold2. Note that OB3 does not exist in Naganishia BRCA2.

Figure S1.. Comparison of the C-terminal region of BRCA2 homologs.

Annotated domains and Dss1 interaction sites are based on the crystal structure of the C-terminal region of mouse BRCA2 (Yang et al, 2002). H. sapiens (P51587), amino acids 2,445–3,227; N. liquefaciens (GHJ86783), amino acids 668–1,193; C. neoformans (UOH82896), amino acids 383–854; U. maydis (XP_011389639), amino acids 503–1,075. Helical, helical domain; tower, tower domain; Dss1 interaction, Dss1 interaction amino acid.

Figure S2.. Comparison of BRC repeats in Naganishia Brh2 and the structures of the C-terminal domain of vertebrate BRCA2.

(A) Comparison of Naganishia BRC2 and BRC3. Shown on the top is previously established BRC consensus. Nl, N. liquefaciens. (B) Comparison between the crystal structure of the mouse BRCA2 C-terminal domain with Dss1 and ssDNA (accession 1MJE) and the structural model of human BRCA2 C-terminus, generated by AlphaFold2. HS, H. sapiens.

Figure 2.. Identification and characterization of Dss1 homologs in N. liquefaciens.

(A) Alignment of Dss1 and its homologs. Hs, H. sapiens; Nl, N. liquefaciens; Cn, C. neoformans; Um, U. maydis. (B) Protein interactions assessed using the yeast two-hybrid assay. DBD, Gal4 DNA-binding domain; GAD, Gal4 transcription activation domain. “Control” indicates synthetic medium lacking tryptophan and leucin. “-HIS, -ADE” indicates a synthetic medium lacking histidine and adenine, and tryptophan and leucin. (C) Monitoring cell growth of various strains. Error bars, SD (n = 3). Source data are available for this figure.

Figure 3.. BRH2 and RAD51 function in the same pathway in DNA damage repair.

(A) UV and (B) γ-ray sensitivity results. Strains used: WT, MP1; rad51∆, MP35; brh2∆, MP31; rad51∆ brh2∆, MP37; rad52∆, MP33; rad51∆ rad52∆, MP39; brh2∆ rad52∆, MP41; rad51∆ brh2∆ rad52∆, MP101. Error bars, SD (n = 3). Source data are available for this figure.

Figure 4.. Brh2 and Rad51 function in the same homologous recombination pathway.

(A) Schematic drawing of the gene targeting system employed. (B) Gene-targeting efficiency with the targeting DNA with 1-kb homology arms. (C) The same as (B) except that the targeting DNA was with 80-bp homologous arms. Strains used are as follows: WT, MP1; rad51∆, MP35; brh2∆, MP31; rad51∆ brh2∆, MP151; rad52∆, MP33; rad51∆ rad52∆, MP112; brh2∆ rad52∆, MP139; ku70∆, MP94; rad51∆ ku70∆, MP108; brh2∆ ku70∆, MP143; rad51∆ brh2∆ ku70∆, MP131; rad52∆ ku70∆, MP75; rad51∆ rad52∆ ku70∆, MP113; brh2∆ rad52∆ ku70∆, MP147. Error bars, SD. n = 3 in all the experiments except strains carrying rad52∆ (n = 9). Statistical significance was tested using unpaired two-tailed t test (ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). Source data are available for this figure.