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1.
Genes Dev ; 37(13-14): 621-639, 2023 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-37541760

RESUMO

Punctuated bursts of structural genomic variations (SVs) have been described in various organisms, but their etiology remains incompletely understood. Homologous recombination (HR) is a template-guided mechanism of repair of DNA double-strand breaks and stalled or collapsed replication forks. We recently identified a DNA break amplification and genome rearrangement pathway originating from the endonucleolytic processing of a multi-invasion (MI) DNA joint molecule formed during HR. Genome-wide approaches confirmed that multi-invasion-induced rearrangement (MIR) frequently leads to several repeat-mediated SVs and aneuploidies. Using molecular and genetic analysis and a novel, highly sensitive proximity ligation-based assay for chromosomal rearrangement quantification, we further delineate two MIR subpathways. MIR1 is a universal pathway occurring in any sequence context, which generates secondary breaks and frequently leads to additional SVs. MIR2 occurs only if recombining donors exhibit substantial homology and results in sequence insertion without additional breaks or SVs. The most detrimental MIR1 pathway occurs late on a subset of persisting DNA joint molecules in a PCNA/Polδ-independent manner, unlike recombinational DNA synthesis. This work provides a refined mechanistic understanding of these HR-based SV formation pathways and shows that complex repeat-mediated SVs can occur without displacement DNA synthesis. Sequence signatures for inferring MIR1 from long-read data are proposed.


Assuntos
Instabilidade Genômica , Rearranjo Gênico , Recombinação Homóloga , Seleção Genética , DNA/genética , DNA/metabolismo , Cromossomos Fúngicos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
2.
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
3.
bioRxiv ; 2023 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-36993162

RESUMO

Punctuated bursts of structural genomic variations (SVs) have been described in various organisms, but their etiology remains incompletely understood. Homologous recombination (HR) is a template-guided mechanism of repair of DNA double-strand breaks and stalled or collapsed replication forks. We recently identified a DNA break amplification and genome rearrangement pathway originating from the endonucleolytic processing of a multi-invasion (MI) DNA joint molecule formed during HR. Genome-wide sequencing approaches confirmed that multi-invasion-induced rearrangement (MIR) frequently leads to several repeat-mediated SVs and aneuploidies. Using molecular and genetic analysis, and a novel, highly sensitive proximity ligation-based assay for chromosomal rearrangement quantification, we further delineate two MIR sub-pathways. MIR1 is a universal pathway occurring in any sequence context, which generates secondary breaks and frequently leads to additional SVs. MIR2 occurs only if recombining donors exhibit substantial homology, and results in sequence insertion without additional break or SV. The most detrimental MIR1 pathway occurs late on a subset of persisting DNA joint molecules in a PCNA/Polδ-independent manner, unlike recombinational DNA synthesis. This work provides a refined mechanistic understanding of these HR-based SV formation pathways and shows that complex repeat-mediated SVs can occur without displacement DNA synthesis. Sequence signatures for inferring MIR1 from long-read data are proposed.

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