Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 7 de 7
Filtrar
1.
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
2.
EMBO Rep ; 21(8): e49823, 2020 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-32558186

RESUMO

The newly identified shieldin complex, composed of SHLD1, SHLD2, SHLD3, and REV7, lies downstream of 53BP1 and acts to inhibit DNA resection and promote NHEJ. Here, we show that Shld2-/- mice have defective class switch recombination (CSR) and that loss of SHLD2 can suppress the embryonic lethality of a Brca1Δ11 mutation, highlighting its role as a key effector of 53BP1. Lymphocyte development and RAG1/2-mediated recombination were unaffected by SHLD2 deficiency. Interestingly, a significant fraction of Shld2-/- primary B-cells and 53BP1- and shieldin-deficient CH12F3-2 B-cells permanently lose expression of immunoglobulin upon induction of CSR; this population of Ig-negative cells is also seen in other NHEJ-deficient cells and to a much lesser extent in WT cells. This loss of Ig is due to recombination coupled with overactive resection and loss of coding exons in the downstream acceptor constant region. Collectively, these data show that SHLD2 is the key effector of 53BP1 and critical for CSR in vivo by suppressing large deletions within the Igh locus.


Assuntos
Quebras de DNA de Cadeia Dupla , Switching de Imunoglobulina , Animais , Switching de Imunoglobulina/genética , Camundongos
3.
Nature ; 560(7716): 117-121, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30022168

RESUMO

53BP1 is a chromatin-binding protein that regulates the repair of DNA double-strand breaks by suppressing the nucleolytic resection of DNA termini1,2. This function of 53BP1 requires interactions with PTIP3 and RIF14-9, the latter of which recruits REV7 (also known as MAD2L2) to break sites10,11. How 53BP1-pathway proteins shield DNA ends is currently unknown, but there are two models that provide the best potential explanation of their action. In one model the 53BP1 complex strengthens the nucleosomal barrier to end-resection nucleases12,13, and in the other 53BP1 recruits effector proteins with end-protection activity. Here we identify a 53BP1 effector complex, shieldin, that includes C20orf196 (also known as SHLD1), FAM35A (SHLD2), CTC-534A2.2 (SHLD3) and REV7. Shieldin localizes to double-strand-break sites in a 53BP1- and RIF1-dependent manner, and its SHLD2 subunit binds to single-stranded DNA via OB-fold domains that are analogous to those of RPA1 and POT1. Loss of shieldin impairs non-homologous end-joining, leads to defective immunoglobulin class switching and causes hyper-resection. Mutations in genes that encode shieldin subunits also cause resistance to poly(ADP-ribose) polymerase inhibition in BRCA1-deficient cells and tumours, owing to restoration of homologous recombination. Finally, we show that binding of single-stranded DNA by SHLD2 is critical for shieldin function, consistent with a model in which shieldin protects DNA ends to mediate 53BP1-dependent DNA repair.


Assuntos
Reparo do DNA , Complexos Multiproteicos/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Animais , Sistemas CRISPR-Cas , Linhagem Celular , Quebras de DNA de Cadeia Dupla , DNA de Cadeia Simples/genética , Feminino , Genes BRCA1 , Humanos , Switching de Imunoglobulina/genética , Camundongos , Modelos Biológicos , Complexos Multiproteicos/química , Complexos Multiproteicos/deficiência , Complexos Multiproteicos/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Proteínas de Ligação a Telômeros/metabolismo , Proteína Supressora de Tumor p53/deficiência
4.
Nat Commun ; 9(1): 1006, 2018 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-29520062

RESUMO

Class switch recombination (CSR) has a fundamental function during humoral immune response and involves the induction and subsequent repair of DNA breaks in the immunoglobulin (Ig) switch regions. Here we show the role of Usp22, the SAGA complex deubiquitinase that removes ubiquitin from H2B-K120, in the repair of programmed DNA breaks in vivo. Ablation of Usp22 in primary B cells results in defects in γH2AX and impairs the classical non-homologous end joining (c-NHEJ), affecting both V(D)J recombination and CSR. Surprisingly, Usp22 depletion causes defects in CSR to various Ig isotypes, but not IgA. We further demonstrate that IgG CSR primarily relies on c-NHEJ, whereas CSR to IgA is more reliant on the alternative end joining pathway, indicating that CSR to different isotypes involves distinct DNA repair pathways. Hence, Usp22 is the first deubiquitinase reported to regulate both V(D)J recombination and CSR in vivo by facilitating c-NHEJ.


Assuntos
Reparo do DNA por Junção de Extremidades , Enzimas Desubiquitinantes/metabolismo , Endopeptidases/metabolismo , Imunidade Humoral/genética , Switching de Imunoglobulina , Recombinação V(D)J , Animais , Linfócitos B , Enzimas Desubiquitinantes/genética , Endopeptidases/genética , Feminino , Histonas/genética , Histonas/metabolismo , Isotipos de Imunoglobulinas/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Cultura Primária de Células , Ubiquitina/metabolismo , Ubiquitina Tiolesterase
5.
Proc Natl Acad Sci U S A ; 115(11): 2800-2805, 2018 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-29472448

RESUMO

Activation-induced cytidine deaminase (AID) inflicts DNA damage at Ig genes to initiate class switch recombination (CSR) and chromosomal translocations. However, the DNA lesions formed during these processes retain an element of randomness, and thus knowledge of the relationship between specific DNA lesions and AID-mediated processes remains incomplete. To identify necessary and sufficient DNA lesions in CSR, the Cas9 endonuclease and nickase variants were used to program DNA lesions at a greater degree of predictability than is achievable with conventional induction of CSR. Here we show that Cas9-mediated nicks separated by up to 250 nucleotides on opposite strands can mediate CSR. Staggered double-stranded breaks (DSBs) result in more end resection and junctional microhomology than blunt DSBs. Moreover, Myc-Igh chromosomal translocations, which are carried out primarily by alternative end joining (A-EJ), were preferentially induced by 5' DSBs. These data indicate that DSBs with 5' overhangs skew intrachromosomal and interchromosomal end-joining toward A-EJ. In addition to lending potential insight to AID-mediated phenomena, this work has broader carryover implications in DNA repair and lymphomagenesis.


Assuntos
Cromossomos de Mamíferos/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Recombinação Genética , Animais , Linfócitos B/citologia , Linfócitos B/metabolismo , Citidina Desaminase/metabolismo , Camundongos , Translocação Genética
6.
Sci Rep ; 6: 37215, 2016 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-27853268

RESUMO

Class switch recombination (CSR) in B cells requires the timely repair of DNA double-stranded breaks (DSBs) that result from lesions produced by activation-induced cytidine deaminase (AID). Through a genome-wide RNAi screen, we identified Kin17 as a gene potentially involved in the maintenance of CSR in murine B cells. In this study, we confirm a critical role for Kin17 in CSR independent of AID activity. Furthermore, we make evident that DSBs generated by AID or ionizing radiation require Kin17 for efficient repair and resolution. Our report shows that reduced Kin17 results in an elevated deletion frequency following AID mutational activity in the switch region. In addition, deficiency in Kin17 affects the functionality of multiple DSB repair pathways, namely homologous recombination, non-homologous end-joining, and alternative end-joining. This report demonstrates the importance of Kin17 as a critical factor that acts prior to the repair phase of DSB repair and is of bona fide importance for CSR.


Assuntos
Linfócitos B/imunologia , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades/imunologia , Proteínas de Ligação a DNA/imunologia , Switching de Imunoglobulina/imunologia , Proteínas de Ligação a RNA/imunologia , Linhagem Celular Tumoral , Reparo do DNA por Junção de Extremidades/genética , Proteínas de Ligação a DNA/genética , Humanos , Switching de Imunoglobulina/genética , Proteínas de Ligação a RNA/genética
7.
Cell Rep ; 15(7): 1554-1565, 2016 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-27160905

RESUMO

Class switch recombination (CSR) requires activation-induced deaminase (AID) to instigate double-stranded DNA breaks at the immunoglobulin locus. DNA breaks activate the DNA damage response (DDR) by inducing phosphorylation of histone H2AX followed by non-homologous end joining (NHEJ) repair. We carried out a genome-wide screen to identify CSR factors. We found that Usp22, Eny2, and Atxn7, members of the Spt-Ada-Gcn5-acetyltransferase (SAGA) deubiquitination module, are required for deubiquitination of H2BK120ub following DNA damage, are critical for CSR, and function downstream of AID. The SAGA deubiquitinase activity was required for optimal irradiation-induced γH2AX formation, and failure to remove H2BK120ub inhibits ATM- and DNAPK-induced γH2AX formation. Consistent with this effect, these proteins were found to function upstream of various double-stranded DNA repair pathways. This report demonstrates that deubiquitination of histone H2B impacts the early stages of the DDR and is required for the DNA repair phase of CSR.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Reparo do DNA , Proteína Quinase Ativada por DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Histonas/metabolismo , Switching de Imunoglobulina/genética , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Ubiquitinação , Animais , Citidina Desaminase , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades/efeitos da radiação , Reparo do DNA/efeitos da radiação , Endopeptidases/metabolismo , Recombinação Homóloga/efeitos da radiação , Camundongos , Interferência de RNA , Radiação Ionizante , Hipermutação Somática de Imunoglobulina/efeitos da radiação , Fatores de Transcrição/metabolismo , Ubiquitina Tiolesterase , Ubiquitinação/efeitos da radiação
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA