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1.
Mol Cell ; 82(24): 4664-4680.e9, 2022 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-36455556

RESUMO

POLQ is a key effector of DSB repair by microhomology-mediated end-joining (MMEJ) and is overexpressed in many cancers. POLQ inhibitors confer synthetic lethality in HR and Shieldin-deficient cancer cells, which has been proposed to reflect a critical dependence on the DSB repair pathway by MMEJ. Whether POLQ also operates independent of MMEJ remains unexplored. Here, we show that POLQ-deficient cells accumulate post-replicative ssDNA gaps upon BRCA1/2 loss or PARP inhibitor treatment. Biochemically, cooperation between POLQ helicase and polymerase activities promotes RPA displacement and ssDNA-gap fill-in, respectively. POLQ is also capable of microhomology-mediated gap skipping (MMGS), which generates deletions during gap repair that resemble the genomic scars prevalent in POLQ overexpressing cancers. Our findings implicate POLQ in mutagenic post-replicative gap sealing, which could drive genome evolution in cancer and whose loss places a critical dependency on HR for gap protection and repair and cellular viability.


Assuntos
Quebras de DNA de Cadeia Dupla , Neoplasias , Humanos , Replicação do DNA/genética , Instabilidade Genômica , DNA de Cadeia Simples/genética , Mutações Sintéticas Letais , Reparo do DNA por Junção de Extremidades , Neoplasias/genética
2.
Mol Cell ; 81(14): 3018-3030.e5, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34102106

RESUMO

Mammalian DNA base excision repair (BER) is accelerated by poly(ADP-ribose) polymerases (PARPs) and the scaffold protein XRCC1. PARPs are sensors that detect single-strand break intermediates, but the critical role of XRCC1 during BER is unknown. Here, we show that protein complexes containing DNA polymerase ß and DNA ligase III that are assembled by XRCC1 prevent excessive engagement and activity of PARP1 during BER. As a result, PARP1 becomes "trapped" on BER intermediates in XRCC1-deficient cells in a manner similar to that induced by PARP inhibitors, including in patient fibroblasts from XRCC1-mutated disease. This excessive PARP1 engagement and trapping renders BER intermediates inaccessible to enzymes such as DNA polymerase ß and impedes their repair. Consequently, PARP1 deletion rescues BER and resistance to base damage in XRCC1-/- cells. These data reveal excessive PARP1 engagement during BER as a threat to genome integrity and identify XRCC1 as an "anti-trapper" that prevents toxic PARP1 activity.


Assuntos
Reparo do DNA/genética , DNA/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/metabolismo , Animais , Linhagem Celular , Quebras de DNA de Cadeia Simples , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , DNA Ligase Dependente de ATP/metabolismo , DNA Polimerase beta/metabolismo , Reparo do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Ligação Proteica/efeitos dos fármacos
3.
Nat Cell Biol ; 23(12): 1287-1298, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34811483

RESUMO

Genetic defects in the repair of DNA single-strand breaks (SSBs) can result in neurological disease triggered by toxic activity of the single-strand-break sensor protein PARP1. However, the mechanism(s) by which this toxic PARP1 activity triggers cellular dysfunction are unclear. Here we show that human cells lacking XRCC1 fail to rapidly recover transcription following DNA base damage, a phenotype also observed in patient-derived fibroblasts with XRCC1 mutations and Xrcc1-/- mouse neurons. This defect is caused by excessive/aberrant PARP1 activity during DNA base excision repair, resulting from the loss of PARP1 regulation by XRCC1. We show that aberrant PARP1 activity suppresses transcriptional recovery during base excision repair by promoting excessive recruitment and activity of the ubiquitin protease USP3, which as a result reduces the level of monoubiquitinated histones important for normal transcriptional regulation. Importantly, inhibition and/or deletion of PARP1 or USP3 restores transcriptional recovery in XRCC1-/- cells, highlighting PARP1 and USP3 as possible therapeutic targets in neurological disease.


Assuntos
Quebras de DNA de Cadeia Simples , Reparo do DNA/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Transcrição Gênica/genética , Proteases Específicas de Ubiquitina/metabolismo , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/metabolismo , Animais , Linhagem Celular Tumoral , DNA/genética , Histonas/metabolismo , Humanos , Peróxido de Hidrogênio/toxicidade , Camundongos , Camundongos Knockout , Estresse Oxidativo/genética , Poli(ADP-Ribose) Polimerase-1/genética , Ubiquitinação/fisiologia , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/genética
4.
J Immunol Sci ; 2(1): 26-31, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29652413

RESUMO

ATM kinase is a master regulator of the DNA damage response (DDR). A recently published report from the d'Adda di Fagagna laboratory1 sheds a light onto our understanding of ATM activation. In this short-commentary we will expand on this and other work to perceive better some of the aspects of ATM regulation.

5.
Mutat Res ; 808: 20-27, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29482073

RESUMO

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) controls one of the most frequently used DNA repair pathways in a cell, the non-homologous end joining (NHEJ) pathway. However, the exact role of DNA-PKcs in NHEJ remains poorly defined. Here we show that NOTCH1 attenuates DNA-PKcs-mediated autophosphorylation, as well as the phosphorylation of its specific substrate XRCC4. Surprisingly, NOTCH1-expressing cells do not display any significant impairment in the DNA damage repair, nor cellular survival, and remain sensitive to small molecule DNA-PKcs inhibitor. Additionally, in vitro DNA-PKcs kinase assay shows that NOTCH1 does not inhibit DNA-PKcs kinase activity, implying that NOTCH1 acts on DNA-PKcs through a different mechanism. Together, our set of results suggests that NOTCH1 is a physiological modulator of DNA-PKcs, and that it can be a useful tool to clarify the mechanisms by which DNA-PKcs governs NHEJ DNA repair.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Reparo do DNA , Proteína Quinase Ativada por DNA/metabolismo , Proteínas Nucleares/metabolismo , Receptor Notch1/metabolismo , Proteína Quinase Ativada por DNA/genética , Células HEK293 , Humanos , Proteínas Nucleares/genética , Fosforilação , Receptor Notch1/genética
6.
Nat Commun ; 9(1): 5376, 2018 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-30560944

RESUMO

DNA double-strand breaks (DSBs) are toxic DNA lesions, which, if not properly repaired, may lead to genomic instability, cell death and senescence. Damage-induced long non-coding RNAs (dilncRNAs) are transcribed from broken DNA ends and contribute to DNA damage response (DDR) signaling. Here we show that dilncRNAs play a role in DSB repair by homologous recombination (HR) by contributing to the recruitment of the HR proteins BRCA1, BRCA2, and RAD51, without affecting DNA-end resection. In S/G2-phase cells, dilncRNAs pair to the resected DNA ends and form DNA:RNA hybrids, which are recognized by BRCA1. We also show that BRCA2 directly interacts with RNase H2, mediates its localization to DSBs in the S/G2 cell-cycle phase, and controls DNA:RNA hybrid levels at DSBs. These results demonstrate that regulated DNA:RNA hybrid levels at DSBs contribute to HR-mediated repair.


Assuntos
Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , RNA Longo não Codificante/metabolismo , Reparo de DNA por Recombinação , Ribonuclease H/metabolismo , Proteína BRCA1/genética , Proteína BRCA2/genética , Linhagem Celular Tumoral , DNA/genética , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Fase G2/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , RNA Longo não Codificante/genética , RNA Interferente Pequeno/metabolismo , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Ribonuclease H/genética , Fase S/genética
7.
Cell Rep ; 16(8): 2068-2076, 2016 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-27524627

RESUMO

The DNA damage response (DDR) signal transduction pathway is responsible for sensing DNA damage and further relaying this signal into the cell. ATM is an apical DDR kinase that orchestrates the activation and the recruitment of downstream DDR factors to induce cell-cycle arrest and repair. We have previously shown that NOTCH1 inhibits ATM activation upon DNA damage, but the underlying mechanism remains unclear. Here, we show that NOTCH1 does not impair ATM recruitment to DNA double-strand breaks (DSBs). Rather, NOTCH1 prevents binding of FOXO3a and KAT5/Tip60 to ATM through a mechanism in which NOTCH1 competes with FOXO3a for ATM binding. Lack of FOXO3a binding to ATM leads to the loss of KAT5/Tip60 association with ATM. Moreover, expression of NOTCH1 or depletion of ATM impairs the formation of the FOXO3a-KAT5/Tip60 protein complex. Finally, we show that pharmacological induction of FOXO3a nuclear localization sensitizes NOTCH1-driven cancers to DNA-damage-induced cell death.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/genética , Reparo do DNA , Proteína Forkhead Box O3/genética , Linfócitos/metabolismo , Lisina Acetiltransferase 5/genética , Receptor Notch1/genética , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Ligação Competitiva , Morte Celular/efeitos dos fármacos , Morte Celular/efeitos da radiação , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Proteína Forkhead Box O3/metabolismo , Raios gama , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Linfócitos/efeitos dos fármacos , Linfócitos/patologia , Linfócitos/efeitos da radiação , Lisina Acetiltransferase 5/metabolismo , Metformina/farmacologia , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Embrionárias Murinas/efeitos da radiação , Ligação Proteica , Receptor Notch1/metabolismo , Transdução de Sinais
8.
Nat Struct Mol Biol ; 22(5): 417-24, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25895060

RESUMO

The DNA-damage response (DDR) ensures genome stability and proper inheritance of genetic information, both of which are essential to survival. It is presently unclear to what extent other signaling pathways modulate DDR function. Here we show that Notch receptor binds and inactivates ATM kinase and that this mechanism is evolutionarily conserved in Caenorhabditis elegans, Xenopus laevis and humans. In C. elegans, the Notch pathway impairs DDR signaling in gonad germ cells. In mammalian cells, activation of human Notch1 leads to reduced ATM signaling in a manner independent of Notch1 transcriptional activity. Notch1 binds directly to the regulatory FATC domain of ATM and inhibits ATM kinase activity. Notch1 and ATM activation are inversely correlated in human breast cancers, and inactivation of ATM by Notch1 contributes to the survival of Notch1-driven leukemia cells upon DNA damage.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Caenorhabditis elegans/metabolismo , Reparo do DNA/genética , Receptor Notch1/metabolismo , Xenopus laevis/metabolismo , Animais , Apoptose/genética , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/genética , Sítios de Ligação , Linhagem Celular Tumoral , Dano ao DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática/genética , Células HEK293 , Células HeLa , Humanos , Complexos Multiproteicos , Neoplasias/genética , Ligação Proteica , Estrutura Terciária de Proteína , Receptor Notch1/antagonistas & inibidores , Receptor Notch1/genética , Transdução de Sinais/genética , Transcrição Gênica/genética
9.
Mech Ageing Dev ; 133(6): 444-55, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22613224

RESUMO

Curcumin, a natural polyphenol derived from the rhizome of Curcuma longa, is a potent anticancer agent, which restricts tumor cell growth both in vitro and in vivo. Thus far curcumin was shown to induce death of cancer cells. This study reports the induction of cellular senescence of human colon cancer cells HCT116 upon curcumin treatment. The SA-ß-galactosidase activation was observed both in p53+/+ and p53-/- cells, however the latter ones were less sensitive to the prosenescent activity of curcumin. Upregulation of p53 and p21 proteins was observed in p53+/+ HCT116, while p53-independent induction of p21 was noticed in p53-/- HCT116. Moreover, the senescence of HCT116 cells was accompanied by autophagy, that was confirmed by electron microscopy observations of autophagosomes in the curcumin-treated cells as well as LC3-II expression, punctue staining of LC3 and increased content of acidic vacuoles. Inhibition of autophagy, due to the diminished expression of ATG5 by RNAi decreased the number of senescent cells induced by curcumin, but did not lead to increased cell death. Altogether, we demonstrated a new antitumor activity of curcumin leading to cancer cell senescence and revealed the presence of a functional link between senescence and autophagy in curcumin-treated cells.


Assuntos
Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Neoplasias Colorretais/patologia , Curcumina/farmacologia , Autofagia/genética , Proteína 5 Relacionada à Autofagia , Proliferação de Células/efeitos dos fármacos , Senescência Celular/genética , Inativação Gênica , Células HCT116 , Humanos , Proteínas Associadas aos Microtúbulos/biossíntese , Proteínas Associadas aos Microtúbulos/genética , Proteína Supressora de Tumor p53/metabolismo , Vacúolos/efeitos dos fármacos , beta-Galactosidase/metabolismo
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