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1.
Nat Commun ; 13(1): 2012, 2022 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-35440629

RESUMO

Transcriptionally active loci are particularly prone to breakage and mounting evidence suggests that DNA Double-Strand Breaks arising in active genes are handled by a dedicated repair pathway, Transcription-Coupled DSB Repair (TC-DSBR), that entails R-loop accumulation and dissolution. Here, we uncover a function for the Bloom RecQ DNA helicase (BLM) in TC-DSBR in human cells. BLM is recruited in a transcription dependent-manner at DSBs where it fosters resection, RAD51 binding and accurate Homologous Recombination repair. However, in an R-loop dissolution-deficient background, we find that BLM promotes cell death. We report that upon excessive RNA:DNA hybrid accumulation, DNA synthesis is enhanced at DSBs, in a manner that depends on BLM and POLD3. Altogether our work unveils a role for BLM at DSBs in active chromatin, and highlights the toxic potential of RNA:DNA hybrids that accumulate at transcription-associated DSBs.


Assuntos
Cromatina , Quebras de DNA de Cadeia Dupla , Cromatina/genética , DNA/genética , DNA/metabolismo , Reparo do DNA , Humanos , RNA/genética , RecQ Helicases/genética , RecQ Helicases/metabolismo , Reparo de DNA por Recombinação
3.
Cancer Res ; 57(5): 828-31, 1997 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-9041180

RESUMO

Although more than 100 different BRCA1 germ-line mutations have already been identified in breast and/or ovarian cancer families, we report for the first time a deleterious genomic rearrangement in BRCA1. A 1-kb deletion comprising exon 17 was found in a large breast and ovarian cancer family, leading to a frameshift in the mutant mRNA due to the absence of exon 17. This deletion is probably the result of a recombination between two closely related Alu sequences. It was not detected by conventional PCR-based methods involving the genomic screening of the 22 coding exons or reverse transcription-PCR because the transcript without exon 17 is unstable in lymphoblastoid cell lines. Therefore, rearrangements in the BRCA1 gene should be sought in breast/ovarian cancer families in which no mutations have been found by PCR-based methods in the coding region or in the splice sites.


Assuntos
Proteína BRCA1/genética , Neoplasias da Mama/genética , Neoplasias Ovarianas/genética , Sequências Repetitivas de Ácido Nucleico , Sequência de Bases , Mapeamento Cromossômico , Éxons , Feminino , Rearranjo Gênico , Humanos , Masculino , Dados de Sequência Molecular , Linhagem , Deleção de Sequência
4.
Cancer Res ; 59(2): 455-61, 1999 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-9927062

RESUMO

Most previous BRCA1 mutation screening studies conducted on breast cancer families were aimed at identifying mutations in the coding sequence and splice sites. Mutations in the promoter and untranslated regions, and large rearrangements are missed by standard mutation detection strategies. To look specifically for such germ-line mutations in the BRCA1 gene, we have analyzed a series of 27 American and 51 French breast cancer families in which no BRCA1 mutation was identified by classical techniques. No mutations were detected in either the promoter or untranslated regions, and we did not find any deletion of the whole gene. Four families were found to carry distinct deletions. Two of them, probably generated by Alu-mediated homologous recombination, were internal deletions of 3 and 23.8 kb, encompassing exon 15 and exons 8-13, respectively. These alterations both lead to a frameshift in the mutant mRNA and to premature stop codon-mediated mRNA decay. The other two deletions encompass exons 1 and 2. On the basis of previous and present analyses, rearrangements represent 8% (3/37) of all mutations in our set of BRCA1 American families. Consequently, the search for rearrangements appears mandatory in BRCA1 mutation screening studies.


Assuntos
Neoplasias da Mama/genética , Deleção de Genes , Rearranjo Gênico , Genes BRCA1 , Mutação em Linhagem Germinativa , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Southern Blotting , Éxons , Feminino , Humanos , Regiões Promotoras Genéticas
5.
Oncogene ; 19(53): 6176-83, 2000 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-11156531

RESUMO

Heritable predisposition to breast and/or ovarian cancer is determined, in part, by germline mutation affecting one of two tumor suppressor genes, BRCA1 and BRCA2 (Miki et al., 1994; Wooster et al., 1995). These genes are required for the maintenance of genomic integrity and for control of homologous recombination in somatic and meiotic cells. Here, we explore the hypothesis that a major role of the BRCA gene products in the somatic DNA damage response centers upon the control of recombination between sister chromatids during S phase. By analogy with model organisms, we suggest that stalling of a mammalian DNA polymerase complex by its encounter with abnormal DNA structure calls forth a series of responses that collaborate to enforce appropriate recombinational outcomes, and to suppress inappropriate or 'illegitimate' recombination.


Assuntos
Proteína BRCA1/genética , Neoplasias da Mama/genética , Proteínas de Ciclo Celular , DNA Polimerase Dirigida por DNA/metabolismo , Proteínas de Neoplasias/genética , Neoplasias Ovarianas/genética , Troca de Cromátide Irmã , Fatores de Transcrição/genética , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Proteína BRCA2 , Reparo do DNA , Replicação do DNA , Células Eucarióticas , Evolução Molecular , Feminino , Humanos , Células Procarióticas , Proteínas Serina-Treonina Quinases/metabolismo , Fase S
6.
Am J Hum Genet ; 60(3): 486-95, 1997 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-9042907

RESUMO

To estimate the proportion of breast cancer families due to BRCA1 or BRCA2, we performed mutation screening of the entire coding regions of both genes supplemented with linkage analysis of 31 families, 8 containing male breast cancers and 23 site-specific female breast cancer. A combination of protein-truncation test and SSCP or heteroduplex analyses was used for mutation screening complemented, where possible, by the analysis of expression level of BRCA1 and BRCA2 alleles. Six of the eight families with male breast cancer revealed frameshift mutations, two in BRCA1 and four in BRCA2. Although most families with female site-specific breast cancers were thought to be due to mutations in either BRCA1 or BRCA2, we identified only eight mutations in our series of 23 site-specific female breast cancer families (34%), four in BRCA1 and four in BRCA2. According to the posterior probabilities calculated for mutation-negative families, based on linkage data and mutation screening results, we would expect 8-10 site-specific female breast cancer families of our series to be due to neither BRCA1 nor BRCA2. Thus, our results suggest the existence of at least one more major breast cancer-susceptibility gene.


Assuntos
Proteína BRCA1/genética , Neoplasias da Mama/genética , Mutação , Proteínas de Neoplasias/genética , Fatores de Transcrição/genética , Proteína BRCA2 , Neoplasias da Mama Masculina/genética , Análise Mutacional de DNA , Feminino , Ligação Genética , Predisposição Genética para Doença , Testes Genéticos , Humanos , Masculino , Linhagem
7.
Genes Chromosomes Cancer ; 31(1): 75-84, 2001 May.
Artigo em Inglês | MEDLINE | ID: mdl-11284038

RESUMO

Genetic linkage data have shown that alterations of the BRCA1 gene are responsible for the majority of hereditary breast and ovarian cancers. BRCA1 germline mutations, however, are found less frequently than expected. Mutation detection strategies, which are generally based on the polymerase chain reaction, therefore focus on point and small gene alterations. These approaches do not allow for the detection of large gene rearrangements, which also can be involved in BRCA1 alterations. Indeed, a few of them, spread over the entire BRCA1 gene, have been detected recently by Southern blotting or transcript analysis. We have developed an alternative strategy allowing a panoramic view of the BRCA1 gene, based on dynamic molecular combing and the design of a full four-color bar code of the BRCA1 region. The strategy was tested with the study of four large BRCA1 rearrangements previously reported. In addition, when screening a series of 10 breast and ovarian cancer families negatively tested for point mutation in BRCA1/2, we found an unreported 17-kb BRCA1 duplication encompassing exons 3 to 8. The detection of rearrangements as small as 2 to 6 kb with respect to the normal size of the studied fragment is achieved when the BRCA1 region is divided into 10 fragments. In addition, as the BRCA1 bar code is a morphologic approach, the direct observation of complex and likely underreported rearrangements, such as inversions and insertions, becomes possible.


Assuntos
DNA de Neoplasias/química , DNA de Neoplasias/genética , Corantes Fluorescentes , Genes BRCA1/genética , Recombinação Genética , Neoplasias da Mama/genética , Deleção Cromossômica , Análise Mutacional de DNA/métodos , Sondas de DNA/genética , DNA de Neoplasias/sangue , Éxons/genética , Feminino , Duplicação Gênica , Humanos , Linfócitos/química , Neoplasias Ovarianas/genética , Células Tumorais Cultivadas
8.
Genes Chromosomes Cancer ; 28(3): 300-7, 2000 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-10862036

RESUMO

Constitutive large deletions and duplications of BRCA1 resulting from Alu-mediated recombination account for a significant proportion of disease-causing mutations in breast and/or ovarian cancer families. Using Southern blot analysis and a protein truncation test (PTT), we have identified a 7.1 kb germline deletion in two families with breast and ovarian cancer. This deletion, which includes exons 8 and 9 and leads to a frameshift at the mRNA level, appears to result from homologous recombination between closely related Alu repeats, one in intron 7 and one in intron 9. In addition to the transcript without exons 8 and 9, analysis of RNA by protein truncation test from individuals with the deletion also identified the presence of alternative splicing of exon 10 from the mutant allele, which results in a transcript that lacks exons 8, 9, and 10. Of interest is that the two American families who carry this deletion are of northern European ancestry and share a common haplotype, suggesting that this deletion may represent a founder mutation. Genes Chromosomes Cancer 28:300-307, 2000.


Assuntos
Processamento Alternativo/genética , Elementos Alu/genética , Proteína BRCA1/genética , Neoplasias da Mama/genética , Deleção Cromossômica , Éxons/genética , Neoplasias Ovarianas/genética , Adulto , DNA de Neoplasias/genética , Feminino , Mutação da Fase de Leitura , Haplótipos , Humanos , Pessoa de Meia-Idade , Recombinação Genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
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