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1.
Nucleic Acids Res ; 47(19): 10151-10165, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31665741

RESUMO

RAD51 plays a central role in homologous recombination during double-strand break repair and in replication fork dynamics. Misregulation of RAD51 is associated with genetic instability and cancer. RAD51 is regulated by many accessory proteins including the highly conserved Shu complex. Here, we report the function of the human Shu complex during replication to regulate RAD51 recruitment to DNA repair foci and, secondly, during replication fork restart following replication fork stalling. Deletion of the Shu complex members, SWS1 and SWSAP1, using CRISPR/Cas9, renders cells specifically sensitive to the replication fork stalling and collapse caused by methyl methanesulfonate and mitomycin C exposure, a delayed and reduced RAD51 response, and fewer sister chromatid exchanges. Our additional analysis identified SPIDR and PDS5B as novel Shu complex interacting partners and genetically function in the same pathway upon DNA damage. Collectively, our study uncovers a protein complex, which consists of SWS1, SWSAP1, SPIDR and PDS5B, involved in DNA repair and provides insight into Shu complex function and composition.


Assuntos
Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação a DNA/genética , Recombinação Homóloga/genética , Proteínas Nucleares/genética , Recombinases Rec A/genética , Fatores de Transcrição/genética , Sistemas CRISPR-Cas/genética , Dano ao DNA/genética , Reparo do DNA/genética , Replicação do DNA/genética , Instabilidade Genômica/genética , Humanos , Complexos Multiproteicos/genética , Rad51 Recombinase/genética , Troca de Cromátide Irmã/genética
2.
Nucleic Acids Res ; 47(19): 10166-10180, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31665745

RESUMO

HORMA domain-containing proteins such as Hop1 play crucial regulatory roles in various chromosomal functions. Here, we investigated roles of the fission yeast Hop1 in the formation of recombination-initiating meiotic DNA double strand breaks (DSBs). Meiotic DSB formation in fission yeast relies on multiple protein-protein interactions such as the one between the chromosome axial protein Rec10 and the DSB-forming complex subunit Rec15. Chromatin immunoprecipitation sequencing demonstrated that Hop1 is colocalized with both Rec10 and Rec15, and we observed physical interactions of Hop1 to Rec15 and Rec10. These results suggest that Hop1 promotes DSB formation by interacting with both axis components and the DSB-forming complex. We also show that Hop1 binding to DSB hotspots requires Rec15 and Rec10, while Hop1 axis binding requires Rec10 only, suggesting that Hop1 is recruited to the axis via Rec10, and to hotspots by hotspot-bound Rec15. Furthermore, we introduced separation-of-function Rec10 mutations, deficient for interaction with either Rec15 or Hop1. These single mutations and hop1Δ conferred only partial defects in meiotic recombination, while the combining the Rec15-binding-deficient rec10 mutation with hop1Δ synergistically reduced meiotic recombination, at least at a model hotspot. Taken together, Hop1 likely functions as a stabilizer for Rec15-Rec10 interaction to promote DSB formation.


Assuntos
Proteínas de Ligação a DNA/genética , Recombinação Homóloga/genética , Proteínas de Schizosaccharomyces pombe/genética , Cromossomos/genética , Quebras de DNA de Cadeia Dupla , Meiose/genética , Mutação , Domínios Proteicos/genética , Schizosaccharomyces/genética , Complexo Sinaptonêmico/genética
3.
PLoS Genet ; 15(10): e1008355, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31584931

RESUMO

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.


Assuntos
Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Recombinação Homóloga/genética , Rad51 Recombinase/genética , Núcleo Celular/genética , Cromátides/genética , Dano ao DNA/genética , Genoma Humano/genética , Células HEK293 , Humanos , Mutação
4.
PLoS Genet ; 15(10): e1008412, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31609962

RESUMO

During meiosis, each chromosome must selectively pair and synapse with its own unique homolog to enable crossover formation and subsequent segregation. How homolog pairing is maintained in early meiosis to ensure synapsis occurs exclusively between homologs is unknown. We aimed to further understand this process by examining the meiotic defects of a unique Drosophila mutant, Mcm5A7. We found that Mcm5A7 mutants are proficient in homolog pairing at meiotic onset yet fail to maintain pairing as meiotic synapsis ensues, causing seemingly normal synapsis between non-homologous loci. This pairing defect corresponds with a reduction of SMC1-dependent centromere clustering at meiotic onset. Overexpressing SMC1 in this mutant significantly restores centromere clustering, homolog pairing, and crossover formation. These data indicate that the initial meiotic pairing of homologs is not sufficient to yield synapsis exclusively between homologs and provide a model in which meiotic homolog pairing must be stabilized by centromeric SMC1 to ensure proper synapsis.


Assuntos
Proteínas de Ciclo Celular/genética , Centrômero/genética , Proteínas Cromossômicas não Histona/genética , Recombinação Homóloga/genética , Meiose/genética , Animais , Pareamento Cromossômico/genética , Segregação de Cromossomos/genética , Drosophila/genética , Complexo Sinaptonêmico , Telômero/genética
5.
Genes Dev ; 33(19-20): 1346-1354, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31575678

RESUMO

The homologous recombination (HR) machinery plays multiple roles in genome maintenance. Best studied in the context of DNA double-stranded break (DSB) repair, recombination enzymes can cleave, pair, and unwind DNA molecules, and collaborate with regulatory proteins to execute multiple DNA processing steps before generating specific repair products. HR proteins also help to cope with problems arising from DNA replication, modulating impaired replication forks or filling DNA gaps. Given these important roles, it is not surprising that each HR step is subject to complex regulation to adjust repair efficiency and outcomes as well as to limit toxic intermediates. Recent studies have revealed intricate regulation of all steps of HR by the protein modifier SUMO, which has been increasingly recognized for its broad influence in nuclear functions. This review aims to connect established roles of SUMO with its newly identified effects on recombinational repair and stimulate further thought on many unanswered questions.


Assuntos
Recombinação Homóloga/genética , Proteína SUMO-1/metabolismo , Animais , Regulação da Expressão Gênica/genética , Humanos , Rad51 Recombinase/metabolismo , Sumoilação
6.
Nat Commun ; 10(1): 4310, 2019 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-31541084

RESUMO

Meiotic crossovers (COs) ensure proper chromosome segregation and redistribute the genetic variation that is transmitted to the next generation. Large populations and the demand for genome-wide, fine-scale resolution challenge existing methods for CO identification. Taking advantage of linked-read sequencing, we develop a highly efficient method for genome-wide identification of COs at kilobase resolution in pooled recombinants. We first test this method using a pool of Arabidopsis F2 recombinants, and recapitulate results obtained from the same plants using individual whole-genome sequencing. By applying this method to a pool of pollen DNA from an F1 plant, we establish a highly accurate CO landscape without generating or sequencing a single recombinant plant. The simplicity of this approach enables the simultaneous generation and analysis of multiple CO landscapes, accelerating the pace at which mechanisms for the regulation of recombination can be elucidated through efficient comparisons of genotypic and environmental effects on recombination.


Assuntos
Genoma de Planta/genética , Técnicas de Genotipagem/métodos , Células Germinativas , Recombinação Homóloga/genética , Recombinação Genética , Arabidopsis/genética , Pontos de Quebra do Cromossomo , Biologia Computacional/métodos , Troca Genética , Metilação de DNA , Genômica , Genótipo , Haplótipos , Pólen/genética , Análise de Sequência de DNA , Sequenciamento Completo do Genoma/métodos
7.
Nat Commun ; 10(1): 4309, 2019 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-31541091

RESUMO

Meiotic recombination rates vary across the genome, often involving localized crossover hotspots and coldspots. Studying the molecular basis and mechanisms underlying this variation has been challenging due to the high cost and effort required to construct individualized genome-wide maps of recombination crossovers. Here we introduce a new method, called ReMIX, to detect crossovers from gamete DNA of a single individual using Illumina sequencing of 10X Genomics linked-read libraries. ReMIX reconstructs haplotypes and identifies the valuable rare molecules spanning crossover breakpoints, allowing quantification of the genomic location and intensity of meiotic recombination. Using a single mouse and stickleback fish, we demonstrate how ReMIX faithfully recovers recombination hotspots and landscapes that have previously been built using hundreds of offspring. ReMIX provides a high-resolution, high-throughput, and low-cost approach to quantify recombination variation across the genome, providing an exciting opportunity to study recombination among multiple individuals in diverse organisms.


Assuntos
Mapeamento Cromossômico/métodos , Biologia Computacional/métodos , Genoma , Recombinação Homóloga/genética , Recombinação Genética , Algoritmos , Animais , Pontos de Quebra do Cromossomo , Troca Genética , Bases de Dados Genéticas , Evolução Molecular , Ligação Genética/genética , Genômica , Haplótipos , Sequenciamento de Nucleotídeos em Larga Escala , Masculino , Meiose , Camundongos , Camundongos Endogâmicos C57BL , Espermatozoides , Baço
8.
Nat Commun ; 10(1): 4388, 2019 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-31558727

RESUMO

Meiosis is a conserved tenet of sexual reproduction in eukaryotes, yet this program is seemingly absent from many extant species. In the human fungal pathogen Candida albicans, mating of diploid cells generates tetraploid products that return to the diploid state via a non-meiotic process of depolyploidization known as concerted chromosome loss (CCL). Here, we report that recombination rates are more than three orders of magnitude higher during CCL than during normal mitotic growth. Furthermore, two conserved 'meiosis-specific' factors play central roles in CCL as SPO11 mediates DNA double-strand break formation while both SPO11 and REC8 regulate chromosome stability and promote inter-homolog recombination. Unexpectedly, SPO11 also promotes DNA repair and recombination during normal mitotic divisions. These results indicate that C. albicans CCL represents a 'parameiosis' that blurs the conventional boundaries between mitosis and meiosis. They also reveal parallels with depolyploidization in mammalian cells and provide potential insights into the evolution of meiosis.


Assuntos
Candida albicans/genética , Diploide , Recombinação Homóloga/genética , Meiose/genética , Tetraploidia , Candidíase/microbiologia , Cromossomos Fúngicos/genética , Dano ao DNA , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Mitose/genética , Recombinases Rec A/genética , Recombinases Rec A/metabolismo , Reparo de DNA por Recombinação
9.
Nucleic Acids Res ; 47(19): 10181-10201, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31495888

RESUMO

Interstrand crosslinks (ICLs) are highly toxic DNA lesions that are repaired via a complex process requiring the coordination of several DNA repair pathways. Defects in ICL repair result in Fanconi anemia, which is characterized by bone marrow failure, developmental abnormalities, and a high incidence of malignancies. SLX4, also known as FANCP, acts as a scaffold protein and coordinates multiple endonucleases that unhook ICLs, resolve homologous recombination intermediates, and perhaps remove unhooked ICLs. In this study, we explored the role of SLX4IP, a constitutive factor in the SLX4 complex, in ICL repair. We found that SLX4IP is a novel regulatory factor; its depletion sensitized cells to treatment with ICL-inducing agents and led to accumulation of cells in the G2/M phase. We further discovered that SLX4IP binds to SLX4 and XPF-ERCC1 simultaneously and that disruption of one interaction also disrupts the other. The binding of SLX4IP to both SLX4 and XPF-ERCC1 not only is vital for maintaining the stability of SLX4IP protein, but also promotes the interaction between SLX4 and XPF-ERCC1, especially after DNA damage. Collectively, these results demonstrate a new regulatory role for SLX4IP in maintaining an efficient SLX4-XPF-ERCC1 complex in ICL repair.


Assuntos
Proteínas de Transporte/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Recombinação Homóloga/genética , Recombinases/genética , DNA/química , DNA/genética , Proteínas de Ligação a DNA/química , Células HEK293 , Humanos , Ligação Proteica/genética
10.
Genes Dev ; 33(17-18): 1175-1190, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31395742

RESUMO

The ribosomal DNA (rDNA) represents a particularly unstable locus undergoing frequent breakage. DNA double-strand breaks (DSBs) within rDNA induce both rDNA transcriptional repression and nucleolar segregation, but the link between the two events remains unclear. Here we found that DSBs induced on rDNA trigger transcriptional repression in a cohesin- and HUSH (human silencing hub) complex-dependent manner throughout the cell cycle. In S/G2 cells, transcriptional repression is further followed by extended resection within the interior of the nucleolus, DSB mobilization at the nucleolar periphery within nucleolar caps, and repair by homologous recombination. We showed that nuclear envelope invaginations frequently connect the nucleolus and that rDNA DSB mobilization, but not transcriptional repression, involves the nuclear envelope-associated LINC complex and the actin pathway. Altogether, our data indicate that rDNA break localization at the nucleolar periphery is not a direct consequence of transcriptional repression but rather is an active process that shares features with the mobilization of persistent DSB in active genes and heterochromatin.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , DNA Ribossômico/genética , Regulação da Expressão Gênica/genética , RNA Longo não Codificante/metabolismo , Nucléolo Celular/metabolismo , Histonas/metabolismo , Recombinação Homóloga/genética , Membrana Nuclear/metabolismo
11.
Genes Dev ; 33(19-20): 1397-1415, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31467087

RESUMO

DNA repair by homologous recombination (HR) is essential for genomic integrity, tumor suppression, and the formation of gametes. HR uses DNA synthesis to repair lesions such as DNA double-strand breaks and stalled DNA replication forks, but despite having a good understanding of the steps leading to homology search and strand invasion, we know much less of the mechanisms that establish recombination-associated DNA polymerization. Here, we report that C17orf53/HROB is an OB-fold-containing factor involved in HR that acts by recruiting the MCM8-MCM9 helicase to sites of DNA damage to promote DNA synthesis. Mice with targeted mutations in Hrob are infertile due to depletion of germ cells and display phenotypes consistent with a prophase I meiotic arrest. The HROB-MCM8-MCM9 pathway acts redundantly with the HELQ helicase, and cells lacking both HROB and HELQ have severely impaired HR, suggesting that they underpin two major routes for the completion of HR downstream from RAD51. The function of HROB in HR is reminiscent of that of gp59, which acts as the replicative helicase loader during bacteriophage T4 recombination-dependent DNA replication. We therefore propose that the loading of MCM8-MCM9 by HROB may similarly be a key step in the establishment of mammalian recombination-associated DNA synthesis.


Assuntos
Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Recombinação Homóloga/genética , Proteínas de Manutenção de Minicromossomo/metabolismo , Animais , Linhagem Celular , DNA Helicases/metabolismo , Feminino , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Infertilidade/genética , Masculino , Camundongos Endogâmicos C57BL , Deleção de Sequência , Células Sf9
12.
Nucleic Acids Res ; 47(17): 9160-9179, 2019 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-31340001

RESUMO

The pleiotropic CCCTC-binding factor (CTCF) plays a role in homologous recombination (HR) repair of DNA double-strand breaks (DSBs). However, the precise mechanistic role of CTCF in HR remains largely unclear. Here, we show that CTCF engages in DNA end resection, which is the initial, crucial step in HR, through its interactions with MRE11 and CtIP. Depletion of CTCF profoundly impairs HR and attenuates CtIP recruitment at DSBs. CTCF physically interacts with MRE11 and CtIP and promotes CtIP recruitment to sites of DNA damage. Subsequently, CTCF facilitates DNA end resection to allow HR, in conjunction with MRE11-CtIP. Notably, the zinc finger domain of CTCF binds to both MRE11 and CtIP and enables proficient CtIP recruitment, DNA end resection and HR. The N-terminus of CTCF is able to bind to only MRE11 and its C-terminus is incapable of binding to MRE11 and CtIP, thereby resulting in compromised CtIP recruitment, DSB resection and HR. Overall, this suggests an important function of CTCF in DNA end resection through the recruitment of CtIP at DSBs. Collectively, our findings identify a critical role of CTCF at the first control point in selecting the HR repair pathway.


Assuntos
Fator de Ligação a CCCTC/genética , Proteínas de Transporte/genética , Recombinação Homóloga/genética , Proteína Homóloga a MRE11/genética , Proteínas Nucleares/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Células HeLa , Humanos , Ligação Proteica/genética , Reparo de DNA por Recombinação/genética , Dedos de Zinco/genética
13.
Nucleic Acids Res ; 47(17): 9132-9143, 2019 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-31329989

RESUMO

Poly(ADP-ribose) polymerases (PARPs) facilitate the repair of DNA single-strand breaks (SSBs). When PARPs are inhibited, unrepaired SSBs colliding with replication forks give rise to cytotoxic double-strand breaks. These are normally rescued by homologous recombination (HR), but, in cells with suboptimal HR, PARP inhibition leads to genomic instability and cell death, a phenomenon currently exploited in the therapy of ovarian cancers in BRCA1/2 mutation carriers. In spite of their promise, resistance to PARP inhibitors (PARPis) has already emerged. In order to identify the possible underlying causes of the resistance, we set out to identify the endogenous source of DNA damage that activates PARPs. We argued that if the toxicity of PARPis is indeed caused by unrepaired SSBs, these breaks must arise spontaneously, because PARPis are used as single agents. We now show that a significant contributor to PARPi toxicity is oxygen metabolism. While BRCA1-depleted or -mutated cells were hypersensitive to the clinically approved PARPi olaparib, its toxicity was significantly attenuated by depletion of OGG1 or MYH DNA glycosylases, as well as by treatment with reactive oxygen species scavengers, growth under hypoxic conditions or chemical OGG1 inhibition. Thus, clinical resistance to PARPi therapy may emerge simply through reduced efficiency of oxidative damage repair.


Assuntos
Proteína BRCA1/genética , DNA Glicosilases/genética , Neoplasias Ovarianas/tratamento farmacológico , Poli(ADP-Ribose) Polimerases/genética , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Simples/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , DNA Glicosilases/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Recombinação Homóloga/efeitos dos fármacos , Recombinação Homóloga/genética , Humanos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Oxirredução/efeitos dos fármacos , Ftalazinas/efeitos adversos , Ftalazinas/farmacologia , Piperazinas/efeitos adversos , Piperazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Mutações Sintéticas Letais/genética
14.
Mutat Res ; 816-818: 111675, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31302572

RESUMO

The accumulation and spatial distribution of 53BP1, BRCA1 and Rad51, key proteins in DNA double-strand break (DSB) repair, was investigated with high temporal resolution over a time span of 24 h, using STED nanoscopy. DNA lesions were induced by irradiation with high-LET (linear energy transfer) α-particles. We show that 53BP1 IRIF formation occurs quickly in almost all cells and after about 6 h the fraction of 53BP1 IRIF positive cells slowly declines. Against the expectations BRCA1 and Rad51 IRIF formation is only shortly delayed but with the maximum of cells showing foci after 6 and 8 h after irradiation. At this stage, almost all IRIF in a given Rad51-positive cell show Rad51 accumulation, suggesting that repair via homologous recombination is attempted at almost all residual DSB sites. The frequency of BRCA1 IRIF positive cells increases much earlier and remains high after Rad51 positive cells start to decline, supporting models claiming that functional roles of BRCA1 change over time. Correlation analysis showed a high degree of correlation of Rad51 with BRCA1, while the exclusion of 53BP1 from the actual resection-zone is demonstrated by anti-correlation of Rad51 and 53BP1. Interestingly, these correlation and anti-correlation patterns exhibit complementary temporal variation.


Assuntos
Proteína BRCA1/genética , Reparo do DNA/genética , DNA/genética , Rad51 Recombinase/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Células HeLa , Recombinação Homóloga/genética , Humanos
15.
Nat Commun ; 10(1): 3190, 2019 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-31320627

RESUMO

Brain metastases (BM) of colorectal cancer (CRC) are rare but lethal, and an understanding of their genomic landscape is lacking. We conduct an analysis of whole-exome sequencing (WES) and whole-genome sequencing (WGS) data on 19 trios of patient-matched BMs, primary CRC tumors, and adjacent normal tissue. Compared with primary CRC, BM exhibits elevated mutational signatures of homologous recombination deficiency (HRD) and mismatch repair deficiency (MMRD). Further analysis reveals two DNA damage response (DDR) signatures could emerge early and are enhanced in BM tissues but are eliminated eventually in matched primary CRC tissues. BM-specific mutations in DDR genes and elevated microsatellite instability (MSI) levels support the importance of DDR in the brain metastasis of CRC. We also identify BM-related genes (e.g., SCN7A, SCN5A, SCN2A, IKZF1, and PDZRN4) that carry frequent BM-specific mutations. These results provide a better understanding of the BM mutational landscape and insights into treatment.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias Encefálicas/genética , Neoplasias Colorretais/genética , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/secundário , Neoplasias Colorretais/patologia , Dano ao DNA/genética , Reparo de Erro de Pareamento de DNA/genética , Recombinação Homóloga/genética , Humanos , Instabilidade de Microssatélites , Mutação/genética , Sequenciamento Completo do Exoma
16.
Nat Commun ; 10(1): 2954, 2019 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-31273204

RESUMO

PARP-1 is rapidly recruited and activated by DNA double-strand breaks (DSBs). Upon activation, PARP-1 synthesizes a structurally complex polymer composed of ADP-ribose units that facilitates local chromatin relaxation and the recruitment of DNA repair factors. Here, we identify a function for PARP-1 in DNA DSB resection. Remarkably, inhibition of PARP-1 leads to hyperresected DNA DSBs. We show that loss of PARP-1 and hyperresection are associated with loss of Ku, 53BP1 and RIF1 resection inhibitors from the break site. DNA curtains analysis show that EXO1-mediated resection is blocked by PARP-1. Furthermore, PARP-1 abrogation leads to increased DNA resection tracks and an increase of homologous recombination in cellulo. Our results, therefore, place PARP-1 activation as a critical early event for DNA DSB repair activation and regulation of resection. Hence, our work has direct implications for the clinical use and effectiveness of PARP inhibition, which is prescribed for the treatment of various malignancies.


Assuntos
Quebras de DNA de Cadeia Dupla , DNA/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Animais , Cromatina/metabolismo , Técnicas de Silenciamento de Genes , Células HeLa , Recombinação Homóloga/genética , Humanos , Camundongos , Modelos Biológicos , Proteínas Nucleares/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Proteínas de Ligação a Telômeros/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo
17.
PLoS Genet ; 15(6): e1008177, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31170160

RESUMO

During meiotic prophase I, double-strand breaks (DSBs) initiate homologous recombination leading to non-crossovers (NCOs) and crossovers (COs). In mouse, 10% of DSBs are designated to become COs, primarily through a pathway dependent on the MLH1-MLH3 heterodimer (MutLγ). Mlh3 contains an endonuclease domain that is critical for resolving COs in yeast. We generated a mouse (Mlh3DN/DN) harboring a mutation within this conserved domain that is predicted to generate a protein that is catalytically inert. Mlh3DN/DN males, like fully null Mlh3-/- males, have no spermatozoa and are infertile, yet spermatocytes have grossly normal DSBs and synapsis events in early prophase I. Unlike Mlh3-/- males, mutation of the endonuclease domain within MLH3 permits normal loading and frequency of MutLγ in pachynema. However, key DSB repair factors (RAD51) and mediators of CO pathway choice (BLM helicase) persist into pachynema in Mlh3DN/DN males, indicating a temporal delay in repair events and revealing a mechanism by which alternative DSB repair pathways may be selected. While Mlh3DN/DN spermatocytes retain only 22% of wildtype chiasmata counts, this frequency is greater than observed in Mlh3-/- males (10%), suggesting that the allele may permit partial endonuclease activity, or that other pathways can generate COs from these MutLγ-defined repair intermediates in Mlh3DN/DN males. Double mutant mice homozygous for the Mlh3DN/DN and Mus81-/- mutations show losses in chiasmata close to those observed in Mlh3-/- males, indicating that the MUS81-EME1-regulated crossover pathway can only partially account for the increased residual chiasmata in Mlh3DN/DN spermatocytes. Our data demonstrate that mouse spermatocytes bearing the MLH1-MLH3DN/DN complex display the proper loading of factors essential for CO resolution (MutSγ, CDK2, HEI10, MutLγ). Despite these functions, mice bearing the Mlh3DN/DN allele show defects in the repair of meiotic recombination intermediates and a loss of most chiasmata.


Assuntos
Proteínas de Ligação a DNA/genética , Endonucleases/genética , Prófase Meiótica I/genética , Proteínas MutL/genética , Animais , Pareamento Cromossômico/genética , Troca Genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Recombinação Homóloga/genética , Masculino , Meiose/genética , Camundongos , Proteína 1 Homóloga a MutL/genética , Proteínas MutS/genética , Rad51 Recombinase/genética , Espermatócitos/crescimento & desenvolvimento , Espermatócitos/metabolismo
18.
Jpn J Clin Oncol ; 49(8): 703-707, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31242303

RESUMO

After a brief summary of the current status of poly-ADP ribose polymerase (PARP) inhibitors for ovarian cancer, we summarize the current status of PARP inhibitors for BRCA wild type ovarian cancer, especially regarding gene alterations other than BRCA, homologous recombination deficiency (HRD), and combinations. Discussion of gene alterations other than BRCA include the results of multiple gene panels studying homologous recombination repair deficiency genes and cancer susceptibility genes, and influences of these alterations on efficacy of PARP inhibitors and cancer susceptibility. Discussions of HRD include the results of phase three trials using HRD assay, the definition of HRD assays, and the latest assays. Discussions of combinations include early phase trial results and ongoing trials combining PARP inhibitors with immune checkpoint inhibitors, anti-angiogenic agents, and triplets.


Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Recombinação Homóloga/genética , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Feminino , Humanos
19.
Nat Commun ; 10(1): 2615, 2019 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-31197154

RESUMO

Balanced expression of multiple genes is central for establishing new biosynthetic pathways or multiprotein cellular complexes. Methods for efficient combinatorial assembly of regulatory sequences (promoters) and protein coding sequences are therefore highly wanted. Here, we report a high-throughput cloning method, called COMPASS for COMbinatorial Pathway ASSembly, for the balanced expression of multiple genes in Saccharomyces cerevisiae. COMPASS employs orthogonal, plant-derived artificial transcription factors (ATFs) and homologous recombination-based cloning for the generation of thousands of individual DNA constructs in parallel. The method relies on a positive selection of correctly assembled pathway variants from both, in vivo and in vitro cloning procedures. To decrease the turnaround time in genomic engineering, COMPASS is equipped with multi-locus CRISPR/Cas9-mediated modification capacity. We demonstrate the application of COMPASS by generating cell libraries producing ß-carotene and co-producing ß-ionone and biosensor-responsive naringenin. COMPASS will have many applications in synthetic biology projects that require gene expression balancing.


Assuntos
Vias Biossintéticas/genética , Engenharia Metabólica/métodos , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Técnicas Biossensoriais/métodos , Sistemas CRISPR-Cas/genética , Clonagem Molecular/métodos , Flavanonas/biossíntese , Recombinação Homóloga/genética , Norisoprenoides/biossíntese , Regiões Promotoras Genéticas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Biologia Sintética/métodos , Fatores de Transcrição/genética , beta Caroteno/biossíntese
20.
Acta Biochim Biophys Sin (Shanghai) ; 51(7): 697-706, 2019 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-31187113

RESUMO

Multiple gene knockouts are often employed in studies of microbial physiology and genetics. However, the selective markers that confer antibiotic resistance are generally limited, so it is necessary to remove these resistance genes before the next round of using, which is time consuming and labor intensive. Here, we created a universal circular gene knockout system for both the gram-negative bacterial Burkholderiales strain DSM 7029 and the gram-positive bacterial Mycobacterium smegmatis mc2 155, by combining the homologous recombination with multiple serine integrase-meditated site-specific recombination systems. In this system, a resistance gene and an integrase gene were constructed within the two attachment sites corresponding to a second, different integrase to form a cassette for gene disruption, which could be easily removed by the second integrase during the subsequent round of gene knockout. The sacB gene was also employed for negative selection. As the integrase-mediated deletion of the resistance/integrase gene cassette was highly efficient and concurrent with the following knockout round, the cyclic use of three cassettes could achieve multiple gene knockout in a sequential manner. Following the modularity concept in synthetic biology, common components of the knockout plasmids were retained as BioBricks, accelerating the knockout plasmids construction process. The circular gene knockout system can also be used for multiple gene insertions and applied to other microorganisms.


Assuntos
Burkholderiales/genética , Resistência Microbiana a Medicamentos/genética , Técnicas de Inativação de Genes/métodos , Genes Bacterianos/genética , Mycobacterium smegmatis/genética , Sítios de Ligação Microbiológicos/genética , Burkholderiales/metabolismo , Recombinação Homóloga/genética , Integrases/genética , Integrases/metabolismo , Mycobacterium smegmatis/metabolismo , Plasmídeos/genética , Reprodutibilidade dos Testes
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