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1.
Proc Natl Acad Sci U S A ; 119(38): e2202727119, 2022 09 20.
Artículo en Inglés | MEDLINE | ID: mdl-36099300

RESUMEN

Mutations in homologous recombination (HR) genes, including BRCA1, BRCA2, and the RAD51 paralog RAD51C, predispose to tumorigenesis and sensitize cancers to DNA-damaging agents and poly(ADP ribose) polymerase inhibitors. However, ∼800 missense variants of unknown significance have been identified for RAD51C alone, impairing cancer risk assessment and therapeutic strategies. Here, we interrogated >50 RAD51C missense variants, finding that mutations in residues conserved with RAD51 strongly predicted HR deficiency and disrupted interactions with other RAD51 paralogs. A cluster of mutations was identified in and around the Walker A box that led to impairments in HR, interactions with three other RAD51 paralogs, binding to single-stranded DNA, and ATP hydrolysis. We generated structural models of the two RAD51 paralog complexes containing RAD51C, RAD51B-RAD51C-RAD51D-XRCC2 and RAD51C-XRCC3. Together with our functional and biochemical analyses, the structural models predict ATP binding at the interface of RAD51C interactions with other RAD51 paralogs, similar to interactions between monomers in RAD51 filaments, and explain the failure of RAD51C variants in binding multiple paralogs. Ovarian cancer patients with variants in this cluster showed exceptionally long survival, which may be relevant to the reversion potential of the variants. This comprehensive analysis provides a framework for RAD51C variant classification. Importantly, it also provides insight into the functioning of the RAD51 paralog complexes.


Asunto(s)
Proteínas de Unión al ADN , Recombinación Homóloga , Neoplasias Ováricas , Recombinasa Rad51 , Proteínas Supresoras de Tumor , Adenosina Trifosfato/metabolismo , Proteínas de Unión al ADN/genética , Femenino , Humanos , Mutación , Neoplasias Ováricas/genética , Recombinasa Rad51/genética , Proteínas Supresoras de Tumor/genética
2.
Nat Commun ; 12(1): 4255, 2021 07 12.
Artículo en Inglés | MEDLINE | ID: mdl-34253720

RESUMEN

Homology-directed repair (HDR), a critical DNA repair pathway in mammalian cells, is complex, leading to multiple outcomes with different impacts on genomic integrity. However, the factors that control these different outcomes are often not well understood. Here we show that SWS1-SWSAP1-SPIDR controls distinct types of HDR. Despite their requirement for stable assembly of RAD51 recombinase at DNA damage sites, these proteins are not essential for intra-chromosomal HDR, providing insight into why patients and mice with mutations are viable. However, SWS1-SWSAP1-SPIDR is critical for inter-homolog HDR, the first mitotic factor identified specifically for this function. Furthermore, SWS1-SWSAP1-SPIDR drives the high level of sister-chromatid exchange, promotes long-range loss of heterozygosity often involved with cancer initiation, and impels the poor growth of BLM helicase-deficient cells. The relevance of these genetic interactions is evident as SWSAP1 loss prolongs Blm-mutant embryo survival, suggesting a possible druggable target for the treatment of Bloom syndrome.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Recombinación Homóloga/genética , Complejos Multiproteicos/metabolismo , Animales , Síndrome de Bloom/genética , Síndrome de Bloom/patología , Proliferación Celular , Células HEK293 , Humanos , Meiosis , Ratones , Mitosis , Células Madre Embrionarias de Ratones/metabolismo , Mutación/genética , Fenotipo , Recombinasa Rad51/metabolismo , Intercambio de Cromátides Hermanas , Análisis de Supervivencia
3.
Artículo en Inglés | MEDLINE | ID: mdl-33832919

RESUMEN

Mutations in homologous recombination (HR) genes predispose to cancer but also sensitize to chemotherapeutics. Although therapy can initially be effective, cancers frequently cease responding, leading to recurrence and poor prognosis. Here we identify a germline mutation in RAD51C, a critical HR factor and known tumor suppressor, in an ovarian cancer patient with exceptionally long, progression-free survival. The RAD51C-T132P mutation is in a highly conserved residue within the nucleotide-binding site and interferes with single-strand DNA binding of the RAD51 paralog complex RAD51B-RAD51C-RAD51D-XRCC2 and association with another RAD51 paralog XRCC3. These biochemical defects lead to highly defective HR and drug sensitivity in tumor cells, ascribing RAD51C-T132P as a deleterious mutation that was likely causal for tumor formation. Conversely, its position within a critical site suggests that it is refractory to secondary mutations that would restore RAD51C gene function and lead to therapy resistance. A need for a greater understanding of the relationship between mutation position and reversion potential of HR genes is underscored, as it may help predict the effectiveness of therapies in patients with HR-deficient cancers.


Asunto(s)
Proteínas de Unión al ADN/genética , Mutación Missense , Neoplasias Ováricas/genética , Animales , Sitios de Unión , Línea Celular , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Resistencia a Antineoplásicos , Femenino , Técnicas de Inactivación de Genes , Mutación de Línea Germinal , Humanos , Insectos , Recombinasa Rad51/genética , Recombinación Genética , Transcriptoma
4.
PLoS Genet ; 15(10): e1008355, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31584931

RESUMEN

Deficiency in several of the classical human RAD51 paralogs [RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3] is associated with cancer predisposition and Fanconi anemia. To investigate their functions, isogenic disruption mutants for each were generated in non-transformed MCF10A mammary epithelial cells and in transformed U2OS and HEK293 cells. In U2OS and HEK293 cells, viable ablated clones were readily isolated for each RAD51 paralog; in contrast, with the exception of RAD51B, RAD51 paralogs are cell-essential in MCF10A cells. Underlining their importance for genomic stability, mutant cell lines display variable growth defects, impaired sister chromatid recombination, reduced levels of stable RAD51 nuclear foci, and hyper-sensitivity to mitomycin C and olaparib, with the weakest phenotypes observed in RAD51B-deficient cells. Altogether these observations underscore the contributions of RAD51 paralogs in diverse DNA repair processes, and demonstrate essential differences in different cell types. Finally, this study will provide useful reagents to analyze patient-derived mutations and to investigate mechanisms of chemotherapeutic resistance deployed by cancers.


Asunto(s)
Reparación del ADN/genética , Proteínas de Unión al ADN/genética , Recombinación Homóloga/genética , Recombinasa Rad51/genética , Núcleo Celular/genética , Cromátides/genética , Daño del ADN/genética , Genoma Humano/genética , Células HEK293 , Humanos , Mutación
5.
Genes (Basel) ; 9(12)2018 Dec 13.
Artículo en Inglés | MEDLINE | ID: mdl-30551670

RESUMEN

The accurate repair of DNA is critical for genome stability and cancer prevention. DNA double-strand breaks are one of the most toxic lesions; however, they can be repaired using homologous recombination. Homologous recombination is a high-fidelity DNA repair pathway that uses a homologous template for repair. One central HR step is RAD51 nucleoprotein filament formation on the single-stranded DNA ends, which is a step required for the homology search and strand invasion steps of HR. RAD51 filament formation is tightly controlled by many positive and negative regulators, which are collectively termed the RAD51 mediators. The RAD51 mediators function to nucleate, elongate, stabilize, and disassemble RAD51 during repair. In model organisms, RAD51 paralogs are RAD51 mediator proteins that structurally resemble RAD51 and promote its HR activity. New functions for the RAD51 paralogs during replication and in RAD51 filament flexibility have recently been uncovered. Mutations in the human RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3, and SWSAP1) are found in a subset of breast and ovarian cancers. Despite their discovery three decades ago, few advances have been made in understanding the function of the human RAD51 paralogs. Here, we discuss the current perspective on the in vivo and in vitro function of the RAD51 paralogs, and their relationship with cancer in vertebrate models.

6.
Cancer Discov ; 7(9): 984-998, 2017 09.
Artículo en Inglés | MEDLINE | ID: mdl-28588062

RESUMEN

High-grade epithelial ovarian carcinomas containing mutated BRCA1 or BRCA2 (BRCA1/2) homologous recombination (HR) genes are sensitive to platinum-based chemotherapy and PARP inhibitors (PARPi), while restoration of HR function due to secondary mutations in BRCA1/2 has been recognized as an important resistance mechanism. We sequenced core HR pathway genes in 12 pairs of pretreatment and postprogression tumor biopsy samples collected from patients in ARIEL2 Part 1, a phase II study of the PARPi rucaparib as treatment for platinum-sensitive, relapsed ovarian carcinoma. In 6 of 12 pretreatment biopsies, a truncation mutation in BRCA1, RAD51C, or RAD51D was identified. In five of six paired postprogression biopsies, one or more secondary mutations restored the open reading frame. Four distinct secondary mutations and spatial heterogeneity were observed for RAD51CIn vitro complementation assays and a patient-derived xenograft, as well as predictive molecular modeling, confirmed that resistance to rucaparib was associated with secondary mutations.Significance: Analyses of primary and secondary mutations in RAD51C and RAD51D provide evidence for these primary mutations in conferring PARPi sensitivity and secondary mutations as a mechanism of acquired PARPi resistance. PARPi resistance due to secondary mutations underpins the need for early delivery of PARPi therapy and for combination strategies. Cancer Discov; 7(9); 984-98. ©2017 AACR.See related commentary by Domchek, p. 937See related article by Quigley et al., p. 999See related article by Goodall et al., p. 1006This article is highlighted in the In This Issue feature, p. 920.


Asunto(s)
Proteínas de Unión al ADN/genética , Resistencia a Antineoplásicos/genética , Indoles/uso terapéutico , Neoplasias Ováricas/tratamiento farmacológico , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Animales , Células CHO , Línea Celular Tumoral , Cricetulus , Femenino , Células HEK293 , Humanos , Mutación , Neoplasias Ováricas/genética
7.
Biochem Cell Biol ; 94(5): 407-418, 2016 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-27224545

RESUMEN

In this review we focus on new insights that challenge our understanding of homologous recombination (HR) and Rad51 regulation. Recent advances using high-resolution microscopy and single molecule techniques have broadened our knowledge of Rad51 filament formation and strand invasion at double-strand break (DSB) sites and at replication forks, which are one of most physiologically relevant forms of HR from yeast to humans. Rad51 filament formation and strand invasion is regulated by many mediator proteins such as the Rad51 paralogues and the Shu complex, consisting of a Shu2/SWS1 family member and additional Rad51 paralogues. Importantly, a novel RAD51 paralogue was discovered in Caenorhabditis elegans, and its in vitro characterization has demonstrated a new function for the worm RAD51 paralogues during HR. Conservation of the human RAD51 paralogues function during HR and repair of replicative damage demonstrate how the RAD51 mediators play a critical role in human health and genomic integrity. Together, these new findings provide a framework for understanding RAD51 and its mediators in DNA repair during multiple cellular contexts.


Asunto(s)
Replicación del ADN , Recombinación Homóloga , Recombinasa Rad51/metabolismo , Animales , Humanos
8.
Genetics ; 203(1): 133-45, 2016 05.
Artículo en Inglés | MEDLINE | ID: mdl-26936927

RESUMEN

Homologous recombination (HR) repairs cytotoxic DNA double-strand breaks (DSBs) with high fidelity. Deficiencies in HR result in genome instability. A key early step in HR is the search for and invasion of a homologous DNA template by a single-stranded RAD-51 nucleoprotein filament. The Shu complex, composed of a SWIM domain-containing protein and its interacting RAD51 paralogs, promotes HR by regulating RAD51 filament dynamics. Despite Shu complex orthologs throughout eukaryotes, our understanding of its function has been most extensively characterized in budding yeast. Evolutionary analysis of the SWIM domain identified Caenorhabditis elegans sws-1 as a putative homolog of the yeast Shu complex member Shu2. Using a CRISPR-induced nonsense allele of sws-1, we show that sws-1 promotes HR in mitotic and meiotic nuclei. sws-1 mutants exhibit sensitivity to DSB-inducing agents and fail to form mitotic RAD-51 foci following treatment with camptothecin. Phenotypic similarities between sws-1 and the two RAD-51 paralogs rfs-1 and rip-1 suggest that they function together. Indeed, we detect direct interaction between SWS-1 and RIP-1 by yeast two-hybrid assay that is mediated by the SWIM domain in SWS-1 and the Walker B motif in RIP-1 Furthermore, RIP-1 bridges an interaction between SWS-1 and RFS-1, suggesting that RIP-1 facilitates complex formation with SWS-1 and RFS-1 We propose that SWS-1, RIP-1, and RFS-1 compose a C. elegans Shu complex. Our work provides a new model for studying Shu complex disruption in the context of a multicellular organism that has important implications as to why mutations in the human RAD51 paralogs are associated with genome instability.


Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Recombinación Homóloga , Recombinasa Rad51/metabolismo , Animales , Sistemas CRISPR-Cas , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas Portadoras/metabolismo , Línea Celular , Expresión Génica , Orden Génico , Sitios Genéticos , Mutación de Línea Germinal , Complejos Multiproteicos/metabolismo , Mutágenos/toxicidad , Mutación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Mapeo de Interacción de Proteínas , Recombinasa Rad51/genética , Reparación del ADN por Recombinación/efectos de los fármacos
9.
J Biol Chem ; 291(9): 4442-52, 2016 Feb 26.
Artículo en Inglés | MEDLINE | ID: mdl-26740628

RESUMEN

DNA damage must be repaired in an accurate and timely fashion to preserve genome stability. Cellular mechanisms preventing genome instability are crucial to human health because genome instability is considered a hallmark of cancer. Collectively referred to as the DNA damage response, conserved pathways ensure proper DNA damage recognition and repair. The function of numerous DNA damage response components is fine-tuned by posttranslational modifications, including ubiquitination. This not only involves the enzyme cascade responsible for conjugating ubiquitin to substrates but also requires enzymes that mediate directed removal of ubiquitin. Deubiquitinases remove ubiquitin from substrates to prevent degradation or to mediate signaling functions. The Saccharomyces cerevisiae deubiquitinase Ubp7 has been characterized previously as an endocytic factor. However, here we identify Ubp7 as a novel factor affecting S phase progression after hydroxyurea treatment and demonstrate an evolutionary and genetic interaction of Ubp7 with DNA damage repair pathways of homologous recombination and nucleotide excision repair. We find that deletion of UBP7 sensitizes cells to hydroxyurea and cisplatin and demonstrate that factors that stabilize replication forks are critical under these conditions. Furthermore, ubp7Δ cells exhibit an S phase progression defect upon checkpoint activation by hydroxyurea treatment. ubp7Δ mutants are epistatic to factors involved in histone maintenance and modification, and we find that a subset of Ubp7 is chromatin-associated. In summary, our results suggest that Ubp7 contributes to S phase progression by affecting the chromatin state at replication forks, and we propose histone H2B ubiquitination as a potential substrate of Ubp7.


Asunto(s)
Cromatina/enzimología , Proteínas Fúngicas/metabolismo , Fase S , Saccharomycetales/enzimología , Proteasas Ubiquitina-Específicas/metabolismo , Cromatina/efectos de los fármacos , Cromatina/metabolismo , Cisplatino/farmacología , Reactivos de Enlaces Cruzados/farmacología , Reparación del ADN , Replicación del ADN/efectos de los fármacos , Proteínas Fúngicas/genética , Eliminación de Gen , Inestabilidad Genómica/efectos de los fármacos , Histonas/metabolismo , Hidroxiurea/farmacología , Viabilidad Microbiana/efectos de los fármacos , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Fase S/efectos de los fármacos , Saccharomycetales/citología , Saccharomycetales/efectos de los fármacos , Saccharomycetales/crecimiento & desarrollo , Proteasas Ubiquitina-Específicas/genética
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