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1.
Mol Cell ; 83(15): 2792-2809.e9, 2023 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-37478847

RESUMO

To maintain genome integrity, cells must accurately duplicate their genome and repair DNA lesions when they occur. To uncover genes that suppress DNA damage in human cells, we undertook flow-cytometry-based CRISPR-Cas9 screens that monitored DNA damage. We identified 160 genes whose mutation caused spontaneous DNA damage, a list enriched in essential genes, highlighting the importance of genomic integrity for cellular fitness. We also identified 227 genes whose mutation caused DNA damage in replication-perturbed cells. Among the genes characterized, we discovered that deoxyribose-phosphate aldolase DERA suppresses DNA damage caused by cytarabine (Ara-C) and that GNB1L, a gene implicated in 22q11.2 syndrome, promotes biogenesis of ATR and related phosphatidylinositol 3-kinase-related kinases (PIKKs). These results implicate defective PIKK biogenesis as a cause of some phenotypes associated with 22q11.2 syndrome. The phenotypic mapping of genes that suppress DNA damage therefore provides a rich resource to probe the cellular pathways that influence genome maintenance.


Assuntos
Sistemas CRISPR-Cas , Dano ao DNA , Humanos , Mutação , Reparo do DNA , Fenótipo
2.
Nat Commun ; 13(1): 957, 2022 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-35177609

RESUMO

The 53BP1-RIF1 pathway antagonizes resection of DNA broken ends and confers PARP inhibitor sensitivity on BRCA1-mutated tumors. However, it is unclear how this pathway suppresses initiation of resection. Here, we identify ASF1 as a partner of RIF1 via an interacting manner similar to its interactions with histone chaperones CAF-1 and HIRA. ASF1 is recruited to distal chromatin flanking DNA breaks by 53BP1-RIF1 and promotes non-homologous end joining (NHEJ) using its histone chaperone activity. Epistasis analysis shows that ASF1 acts in the same NHEJ pathway as RIF1, but via a parallel pathway with the shieldin complex, which suppresses resection after initiation. Moreover, defects in end resection and homologous recombination (HR) in BRCA1-deficient cells are largely suppressed by ASF1 deficiency. Mechanistically, ASF1 compacts adjacent chromatin by heterochromatinization to protect broken DNA ends from BRCA1-mediated resection. Taken together, our findings identify a RIF1-ASF1 histone chaperone complex that promotes changes in high-order chromatin structure to stimulate the NHEJ pathway for DSB repair.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Reparo do DNA por Junção de Extremidades , Chaperonas Moleculares/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Animais , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Galinhas , Cromatina/genética , Epistasia Genética , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Chaperonas Moleculares/genética , Proteínas de Ligação a Telômeros/genética
3.
Cell ; 182(2): 481-496.e21, 2020 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-32649862

RESUMO

The response to DNA damage is critical for cellular homeostasis, tumor suppression, immunity, and gametogenesis. In order to provide an unbiased and global view of the DNA damage response in human cells, we undertook 31 CRISPR-Cas9 screens against 27 genotoxic agents in the retinal pigment epithelium-1 (RPE1) cell line. These screens identified 890 genes whose loss causes either sensitivity or resistance to DNA-damaging agents. Mining this dataset, we discovered that ERCC6L2 (which is mutated in a bone-marrow failure syndrome) codes for a canonical non-homologous end-joining pathway factor, that the RNA polymerase II component ELOF1 modulates the response to transcription-blocking agents, and that the cytotoxicity of the G-quadruplex ligand pyridostatin involves trapping topoisomerase II on DNA. This map of the DNA damage response provides a rich resource to study this fundamental cellular system and has implications for the development and use of genotoxic agents in cancer therapy.


Assuntos
Dano ao DNA , Redes Reguladoras de Genes/fisiologia , Aminoquinolinas/farmacologia , Animais , Sistemas CRISPR-Cas/genética , Linhagem Celular , Citocromo-B(5) Redutase/genética , Citocromo-B(5) Redutase/metabolismo , Dano ao DNA/efeitos dos fármacos , DNA Helicases/genética , DNA Helicases/metabolismo , Reparo do DNA , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Humanos , Camundongos , Ácidos Picolínicos/farmacologia , RNA Guia de Cinetoplastídeos/metabolismo , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética
4.
Mikrochim Acta ; 185(12): 535, 2018 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-30406298

RESUMO

An aptamer based colorimetric assay is described for the determination of zearalenone (ZEN). It is based on the inhibition of the peroxidase-mimicking activity of gold nanoparticles (AuNPs) by the ZEN aptamer. However, in the presence of ZEN, the aptamer is bound by ZEN and can no longer inhibit the peroxidase-mimicking activity of AuNPs. The color change of solution is related to ZEN concentration and observed with bare eyes. Under optimal conditions, the absorbance (at 630 nm) increases linearly in the ZEN concentration range of 10-250 ng·mL-1, and the limit of detection is 10 ng·mL-1. The specificity of the assay was verified by studying the effect of potential interferents. The recoveries from ZEN spiked corn and corn oil range from 92 to 110%, and the relative standard deviations are between 2.4 and 6.4%. The results are in good agreement with those obtained by an ELISA. Graphical abstract Schematic presentation of colorimetric assay for rapid and sensitive determination of zearalenone (ZEN) based on the inhibition of ZEN aptamer on the the peroxidase-like activity of gold nanoparticle (AuNPs).


Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Materiais Biomiméticos/química , Colorimetria/métodos , Ouro/química , Nanopartículas Metálicas/química , Peroxidase/metabolismo , Zearalenona/análise , Aptâmeros de Nucleotídeos/química , Zea mays/química , Zearalenona/metabolismo
5.
Nat Commun ; 9(1): 3925, 2018 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-30254264

RESUMO

53BP1 with its downstream proteins, RIF1, PTIP and REV7, antagonizes BRCA1-dependent homologous recombination (HR) and promotes non-homologous end joining (NHEJ) in an unclear manner. Here we show that REV7 forms a complex with two proteins, FAM35A and C20ORF196. We demonstrate that FAM35A preferentially binds single-strand DNA (ssDNA) in vitro, and is recruited to DSBs as a complex with C20ORF196 and REV7 downstream of RIF1 in vivo. Epistasis analysis shows that both proteins act in the same pathway as RIF1 in NHEJ. The defects in HR pathway to repair DSBs and the reduction in resection of broken DNA ends in BRCA1-mutant cells can be largely suppressed by inactivating FAM35A or C20ORF196, indicating that FAM35A and C20ORF196 prevent end resection in these cells. Together, our data identified a REV7-FAM35A-C20ORF196 complex that binds and protects broken DNA ends to promote the NHEJ pathway for DSB repair.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Complexos Multiproteicos/metabolismo , Transdução de Sinais , Animais , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Proteínas de Ligação a DNA , Células HCT116 , Recombinação Homóloga , Humanos , Proteínas Mad2/genética , Proteínas Mad2/metabolismo , Complexos Multiproteicos/genética , Proteínas/genética , Proteínas/metabolismo , Interferência de RNA , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo
6.
J Biol Chem ; 291(42): 21956-21962, 2016 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-27601467

RESUMO

The replication protein A (RPA) complex binds single-stranded DNA generated at stalled replication forks and recruits other DNA repair proteins to promote recovery of these forks. Here, we identify Ewing tumor-associated antigen 1 (ETAA1), which has been linked to susceptibility to pancreatic cancer, as a new repair protein that is recruited to stalled forks by RPA. We demonstrate that ETAA1 interacts with RPA through two regions, each of which resembles two previously identified RPA-binding domains, RPA70N-binding motif and RPA32C-binding motif, respectively. In response to replication stress, ETAA1 is recruited to stalled forks where it colocalizes with RPA, and this recruitment is diminished when RPA is depleted. Notably, inactivation of the ETAA1 gene increases the collapse level of the stalled replication forks and decreases the recovery efficiency of these forks. Moreover, epistasis analysis shows that ETAA1 stabilizes stalled replication forks in an ataxia telangiectasia and Rad3-related protein (ATR)-independent manner. Thus, our results reveal that ETAA1 is a novel RPA-interacting protein that promotes restart of stalled replication forks.


Assuntos
Antígenos de Superfície/metabolismo , Epistasia Genética/fisiologia , Proteína de Replicação A/metabolismo , Motivos de Aminoácidos , Antígenos de Superfície/genética , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Células HeLa , Humanos , Domínios Proteicos , Proteína de Replicação A/genética
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