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1.
Nucleic Acids Res ; 52(12): 6964-6976, 2024 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-38142462

RESUMO

BRCA2 tumor suppressor protein ensures genome integrity by mediating DNA repair via homologous recombination (HR). This function is executed in part by its canonical DNA binding domain located at the C-terminus (BRCA2CTD), the only folded domain of the protein. Most germline pathogenic missense variants are located in this highly conserved region which binds to single-stranded DNA (ssDNA) and to the acidic protein DSS1. These interactions are essential for the HR function of BRCA2. Here, we report that the variant R2645G, identified in breast cancer and located at the DSS1 interface, unexpectedly increases the ssDNA binding activity of BRCA2CTDin vitro. Human cells expressing this variant display a hyper-recombination phenotype, chromosomal instability in the form of chromatid gaps when exposed to DNA damage, and increased PARP inhibitor sensitivity. In mouse embryonic stem cells (mES), this variant alters viability and confers sensitivity to cisplatin and Mitomycin C. These results suggest that BRCA2 interaction with ssDNA needs to be tightly regulated to limit HR and prevent chromosomal instability and we propose that this control mechanism involves DSS1. Given that several missense variants located within this region have been identified in breast cancer patients, these findings might have clinical implications for carriers.


Assuntos
Proteína BRCA2 , DNA de Cadeia Simples , Ligação Proteica , Humanos , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Animais , Camundongos , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/genética , Instabilidade Cromossômica , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Cisplatino/farmacologia , Dano ao DNA , Mutação de Sentido Incorreto , Feminino , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Células-Tronco Embrionárias Murinas/metabolismo , Linhagem Celular Tumoral , Mitomicina/farmacologia , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Complexo de Endopeptidases do Proteassoma
2.
Nat Commun ; 14(1): 446, 2023 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-36707518

RESUMO

Replication stress (RS) is a major source of genomic instability and is intrinsic to cancer cells. RS is also the consequence of chemotherapeutic drugs for treating cancer. However, adaptation to RS is also a mechanism of resistance to chemotherapy. BRCA2 deficiency results in replication stress in human cells. BRCA2 protein's main functions include DNA repair by homologous recombination (HR) both at induced DNA double-strand breaks (DSB) and spontaneous replicative lesions. At stalled replication forks, BRCA2 protects the DNA from aberrant nucleolytic degradation and is thought to limit the appearance of ssDNA gaps by arresting replication and via post-replicative HR. However, whether and how BRCA2 acts to limit the formation of ssDNA gaps or mediate their repair, remains ill-defined. Here, we use breast cancer variants affecting different domains of BRCA2 to shed light on this function. We demonstrate that the N-terminal DNA binding domain (NTD), and specifically, its dsDNA binding activity, is required to prevent and repair/fill-in ssDNA gaps upon nucleotide depletion but not to limit PARPi-induced ssDNA gaps. Thus, these findings suggest that nucleotide depletion and PARPi trigger gaps via distinct mechanisms and that the NTD of BRCA2 prevents nucleotide depletion-induced ssDNA gaps.


Assuntos
Proteína BRCA2 , Replicação do DNA , Humanos , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Reparo do DNA , DNA/metabolismo , DNA de Cadeia Simples/genética , Nucleotídeos
3.
Cell Death Dis ; 14(11): 753, 2023 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-37980415

RESUMO

Pathogenic variants in BRCA2 are known to significantly increase the lifetime risk of developing breast and ovarian cancers. Sequencing-based genetic testing has resulted in the identification of thousands of BRCA2 variants that are considered to be variants of uncertain significance (VUS) because the disease risk associated with them is unknown. One such variant is p.Arg3052Gln, which has conflicting interpretations of pathogenicity in the ClinVar variant database. Arginine at position 3052 in BRCA2 plays an important role in stabilizing its C-terminal DNA binding domain. We have generated a knock-in mouse model expressing this variant to examine its role on growth and survival in vivo. Homozygous as well as hemizygous mutant mice are viable, fertile and exhibit no overt phenotype. While we did not observe any hematopoietic defects in adults, we did observe a marked reduction in the in vitro proliferative ability of fetal liver cells that were also hypersensitive to PARP inhibitor, olaparib. In vitro studies performed on embryonic and adult fibroblasts derived from the mutant mice showed significant reduction in radiation induced RAD51 foci formation as well as increased genomic instability after mitomycin C treatment. We observed mis-localization of a fraction of R3052Q BRCA2 protein to the cytoplasm which may explain the observed in vitro phenotypes. Our findings suggest that BRCA2 R3052Q should be considered as a hypomorphic variant.


Assuntos
Antineoplásicos , Neoplasias da Mama , Neoplasias Ovarianas , Humanos , Feminino , Camundongos , Animais , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Testes Genéticos , Neoplasias Ovarianas/genética , Homozigoto , Neoplasias da Mama/genética , Proteína BRCA1/genética , Predisposição Genética para Doença
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