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1.
Cell ; 187(4): 861-881.e32, 2024 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-38301646

RESUMO

Genomic instability can trigger cancer-intrinsic innate immune responses that promote tumor rejection. However, cancer cells often evade these responses by overexpressing immune checkpoint regulators, such as PD-L1. Here, we identify the SNF2-family DNA translocase SMARCAL1 as a factor that favors tumor immune evasion by a dual mechanism involving both the suppression of innate immune signaling and the induction of PD-L1-mediated immune checkpoint responses. Mechanistically, SMARCAL1 limits endogenous DNA damage, thereby suppressing cGAS-STING-dependent signaling during cancer cell growth. Simultaneously, it cooperates with the AP-1 family member JUN to maintain chromatin accessibility at a PD-L1 transcriptional regulatory element, thereby promoting PD-L1 expression in cancer cells. SMARCAL1 loss hinders the ability of tumor cells to induce PD-L1 in response to genomic instability, enhances anti-tumor immune responses and sensitizes tumors to immune checkpoint blockade in a mouse melanoma model. Collectively, these studies uncover SMARCAL1 as a promising target for cancer immunotherapy.


Assuntos
Antígeno B7-H1 , DNA Helicases , Imunidade Inata , Melanoma , Evasão Tumoral , Animais , Camundongos , Antígeno B7-H1/metabolismo , Instabilidade Genômica , Melanoma/imunologia , Melanoma/metabolismo , DNA Helicases/metabolismo
2.
Cell ; 184(4): 1081-1097.e19, 2021 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-33606978

RESUMO

Mutations in DNA damage response (DDR) genes endanger genome integrity and predispose to cancer and genetic disorders. Here, using CRISPR-dependent cytosine base editing screens, we identify > 2,000 sgRNAs that generate nucleotide variants in 86 DDR genes, resulting in altered cellular fitness upon DNA damage. Among those variants, we discover loss- and gain-of-function mutants in the Tudor domain of the DDR regulator 53BP1 that define a non-canonical surface required for binding the deubiquitinase USP28. Moreover, we characterize variants of the TRAIP ubiquitin ligase that define a domain, whose loss renders cells resistant to topoisomerase I inhibition. Finally, we identify mutations in the ATM kinase with opposing genome stability phenotypes and loss-of-function mutations in the CHK2 kinase previously categorized as variants of uncertain significance for breast cancer. We anticipate that this resource will enable the discovery of additional DDR gene functions and expedite studies of DDR variants in human disease.


Assuntos
Dano ao DNA , Edição de Genes , Testes Genéticos , Sequência de Aminoácidos , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Sequência de Bases , Sistemas CRISPR-Cas/genética , Camptotecina/farmacologia , Linhagem Celular , Dano ao DNA/genética , Reparo do DNA/genética , Feminino , Humanos , Mutação/genética , Fenótipo , Ligação Proteica , Domínios Proteicos , RNA Guia de Cinetoplastídeos/genética , Inibidores da Topoisomerase/farmacologia , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/química , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Ubiquitina Tiolesterase/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
3.
Mol Cell ; 81(19): 4008-4025.e7, 2021 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-34508659

RESUMO

BRCA1/2 mutant tumor cells display an elevated mutation burden, the etiology of which remains unclear. Here, we report that these cells accumulate ssDNA gaps and spontaneous mutations during unperturbed DNA replication due to repriming by the DNA primase-polymerase PRIMPOL. Gap accumulation requires the DNA glycosylase SMUG1 and is exacerbated by depletion of the translesion synthesis (TLS) factor RAD18 or inhibition of the error-prone TLS polymerase complex REV1-Polζ by the small molecule JH-RE-06. JH-RE-06 treatment of BRCA1/2-deficient cells results in reduced mutation rates and PRIMPOL- and SMUG1-dependent loss of viability. Through cellular and animal studies, we demonstrate that JH-RE-06 is preferentially toxic toward HR-deficient cancer cells. Furthermore, JH-RE-06 remains effective toward PARP inhibitor (PARPi)-resistant BRCA1 mutant cells and displays additive toxicity with crosslinking agents or PARPi. Collectively, these studies identify a protective and mutagenic role for REV1-Polζ in BRCA1/2 mutant cells and provide the rationale for using REV1-Polζ inhibitors to treat BRCA1/2 mutant tumors.


Assuntos
Quebras de DNA de Cadeia Simples , DNA Primase/metabolismo , Replicação do DNA , DNA de Neoplasias/biossíntese , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Enzimas Multifuncionais/metabolismo , Neoplasias/enzimologia , Nucleotidiltransferases/metabolismo , Reparo de DNA por Recombinação , Animais , Antineoplásicos/farmacologia , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Linhagem Celular Tumoral , DNA Primase/genética , DNA de Neoplasias/genética , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , DNA Polimerase Dirigida por DNA/genética , Feminino , Células HEK293 , Humanos , Camundongos Nus , Enzimas Multifuncionais/genética , Mutação , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Inibidores da Síntese de Ácido Nucleico/farmacologia , Nucleotidiltransferases/antagonistas & inibidores , Nucleotidiltransferases/genética , Uracila-DNA Glicosidase/genética , Uracila-DNA Glicosidase/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
4.
PLoS Genet ; 18(11): e1010495, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36374936

RESUMO

Homologous recombination (HR) plays an essential role in the maintenance of genome stability by promoting the repair of cytotoxic DNA double strand breaks (DSBs). More recently, the HR pathway has emerged as a core component of the response to replication stress, in part by protecting stalled replication forks from nucleolytic degradation. In that regard, the mammalian RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3) have been involved in both HR-mediated DNA repair and collapsed replication fork resolution. Still, it remains largely obscure how they participate in both processes, thereby maintaining genome stability and preventing cancer development. To gain better insight into their contribution in cellulo, we mapped the proximal interactome of the classical RAD51 paralogs using the BioID approach. Aside from identifying the well-established BCDX2 and CX3 sub-complexes, the spliceosome machinery emerged as an integral component of our proximal mapping, suggesting a crosstalk between this pathway and the RAD51 paralogs. Furthermore, we noticed that factors involved RNA metabolic pathways are significantly modulated within the BioID of the classical RAD51 paralogs upon exposure to hydroxyurea (HU), pointing towards a direct contribution of RNA processing during replication stress. Importantly, several members of these pathways have prognostic potential in breast cancer (BC), where their RNA expression correlates with poorer patient outcome. Collectively, this study uncovers novel functionally relevant partners of the different RAD51 paralogs in the maintenance of genome stability that could be used as biomarkers for the prognosis of BC.


Assuntos
Instabilidade Genômica , Rad51 Recombinase , Animais , Humanos , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Instabilidade Genômica/genética , Recombinação Homóloga/genética , Quebras de DNA de Cadeia Dupla , RNA , Reparo do DNA/genética , Mamíferos/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo
5.
Mol Cell ; 64(1): 51-64, 2016 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-27546791

RESUMO

The tumor suppressor protein 53BP1, a pivotal regulator of DNA double-strand break (DSB) repair, was first identified as a p53-interacting protein over two decades ago. However, its direct contributions to p53-dependent cellular activities remain undefined. Here, we reveal that 53BP1 stimulates genome-wide p53-dependent gene transactivation and repression events in response to ionizing radiation (IR) and synthetic p53 activation. 53BP1-dependent p53 modulation requires both auto-oligomerization and tandem-BRCT domain-mediated bivalent interactions with p53 and the ubiquitin-specific protease USP28. Loss of these activities results in inefficient p53-dependent cell-cycle checkpoint and exit responses. Furthermore, we demonstrate 53BP1-USP28 cooperation to be essential for normal p53-promoter element interactions and gene transactivation-associated events, yet dispensable for 53BP1-dependent DSB repair regulation. Collectively, our data provide a mechanistic explanation for 53BP1-p53 cooperation in controlling anti-tumorigenic cell-fate decisions and reveal these activities to be distinct and separable from 53BP1's regulation of DNA double-strand break repair pathway choice.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteína Supressora de Tumor p53/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Ubiquitina Tiolesterase/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação , Proteína 9 Associada à CRISPR , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Endonucleases/genética , Endonucleases/metabolismo , Raios gama , Edição de Genes , Regulação da Expressão Gênica , Humanos , Células MCF-7 , Regiões Promotoras Genéticas , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/química , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Ubiquitina Tiolesterase/química , Ubiquitina Tiolesterase/metabolismo
6.
DNA Repair (Amst) ; 95: 102943, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32971328

RESUMO

Over the course of DNA replication, DNA lesions, transcriptional intermediates and protein-DNA complexes can impair the progression of replication forks, thus resulting in replication stress. Failure to maintain replication fork integrity in response to replication stress leads to genomic instability and predisposes to the development of cancer and other genetic disorders. Multiple DNA damage and repair pathways have evolved to allow completion of DNA replication following replication stress, thus preserving genomic integrity. One of the processes commonly induced in response to replication stress is fork reversal, which consists in the remodeling of stalled replication forks into four-way DNA junctions. In normal conditions, fork reversal slows down replication fork progression to ensure accurate repair of DNA lesions and facilitates replication fork restart once the DNA lesions have been removed. However, in certain pathological situations, such as the deficiency of DNA repair factors that protect regressed forks from nuclease-mediated degradation, fork reversal can cause genomic instability. In this review, we describe the complex molecular mechanisms regulating fork reversal, with a focus on the role of the SNF2-family fork remodelers SMARCAL1, ZRANB3 and HLTF, and highlight the implications of fork reversal for tumorigenesis and cancer therapy.


Assuntos
DNA Helicases/metabolismo , Reparo do DNA , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/metabolismo , DNA/metabolismo , Instabilidade Genômica , Humanos
7.
Nat Commun ; 11(1): 2948, 2020 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-32528060

RESUMO

Homologous recombination (HR) mediates the error-free repair of DNA double-strand breaks to maintain genomic stability. Here we characterize C17orf53/MCM8IP, an OB-fold containing protein that binds ssDNA, as a DNA repair factor involved in HR. MCM8IP-deficient cells exhibit HR defects, especially in long-tract gene conversion, occurring downstream of RAD51 loading, consistent with a role for MCM8IP in HR-dependent DNA synthesis. Moreover, loss of MCM8IP confers cellular sensitivity to crosslinking agents and PARP inhibition. Importantly, we report that MCM8IP directly associates with MCM8-9, a helicase complex mutated in primary ovarian insufficiency, and RPA1. We additionally show that the interactions of MCM8IP with MCM8-9 and RPA facilitate HR and promote replication fork progression and cellular viability in response to treatment with crosslinking agents. Mechanistically, MCM8IP stimulates the helicase activity of MCM8-9. Collectively, our work identifies MCM8IP as a key regulator of MCM8-9-dependent DNA synthesis during DNA recombination and replication.


Assuntos
Dano ao DNA , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas de Manutenção de Minicromossomo/metabolismo , Reparo de DNA por Recombinação , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Cromatina/genética , Cromatina/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/genética , Células HCT116 , Células HEK293 , Humanos , Proteínas de Manutenção de Minicromossomo/genética , Mutação , Ligação Proteica , Rad51 Recombinase/metabolismo , Proteína de Replicação A/genética , Proteína de Replicação A/metabolismo
8.
Nat Commun ; 10(1): 3395, 2019 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-31363085

RESUMO

Precise editing of genomic DNA can be achieved upon repair of CRISPR-induced DNA double-stranded breaks (DSBs) by homology-directed repair (HDR). However, the efficiency of this process is limited by DSB repair pathways competing with HDR, such as non-homologous end joining (NHEJ). Here we individually express in human cells 204 open reading frames involved in the DNA damage response (DDR) and determine their impact on CRISPR-mediated HDR. From these studies, we identify RAD18 as a stimulator of CRISPR-mediated HDR. By defining the RAD18 domains required to promote HDR, we derive an enhanced RAD18 variant (e18) that stimulates CRISPR-mediated HDR in multiple human cell types, including embryonic stem cells. Mechanistically, e18 induces HDR by suppressing the localization of the NHEJ-promoting factor 53BP1 to DSBs. Altogether, this study identifies e18 as an enhancer of CRISPR-mediated HDR and highlights the promise of engineering DDR factors to augment the efficiency of precision genome editing.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Dano ao DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Edição de Genes , Humanos , Domínios Proteicos , Engenharia de Proteínas , Reparo de DNA por Recombinação , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética
9.
Nat Commun ; 9(1): 5406, 2018 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-30559443

RESUMO

53BP1 controls a specialized non-homologous end joining (NHEJ) pathway that is essential for adaptive immunity, yet oncogenic in BRCA1 mutant cancers. Intra-chromosomal DNA double-strand break (DSB) joining events during immunoglobulin class switch recombination (CSR) require 53BP1. However, in BRCA1 mutant cells, 53BP1 blocks homologous recombination (HR) and promotes toxic NHEJ, resulting in genomic instability. Here, we identify the protein dimerization hub-DYNLL1-as an organizer of multimeric 53BP1 complexes. DYNLL1 binding stimulates 53BP1 oligomerization, and promotes 53BP1's recruitment to, and interaction with, DSB-associated chromatin. Consequently, DYNLL1 regulates 53BP1-dependent NHEJ: CSR is compromised upon deletion of Dynll1 or its transcriptional regulator Asciz, or by mutation of DYNLL1 binding motifs in 53BP1; furthermore, Brca1 mutant cells and tumours are rendered resistant to poly-ADP ribose polymerase (PARP) inhibitor treatments upon deletion of Dynll1 or Asciz. Thus, our results reveal a mechanism that regulates 53BP1-dependent NHEJ and the therapeutic response of BRCA1-deficient cancers.


Assuntos
Proteína BRCA1/genética , Dineínas do Citoplasma/metabolismo , Reparo do DNA por Junção de Extremidades/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Fatores de Transcrição/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Feminino , Instabilidade Genômica/genética , Células HEK293 , Humanos , Células MCF-7 , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
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