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1.
Mol Cell ; 72(3): 568-582.e6, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30344097

RESUMO

Protecting stalled DNA replication forks from degradation by promiscuous nucleases is essential to prevent genomic instability, a major driving force of tumorigenesis. Several proteins commonly associated with the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) have been implicated in the stabilization of stalled forks. Human CtIP, in conjunction with the MRE11 nuclease complex, plays an important role in HR by promoting DSB resection. Here, we report an unanticipated function for CtIP in protecting reversed forks from degradation. Unlike BRCA proteins, which defend nascent DNA strands from nucleolytic attack by MRE11, we find that CtIP protects perturbed forks from erroneous over-resection by DNA2. Finally, we uncover functionally synergistic effects between CtIP and BRCA1 in mitigating replication-stress-induced genomic instability. Collectively, our findings reveal a DSB-resection- and MRE11-independent role for CtIP in preserving fork integrity that contributes to the survival of BRCA1-deficient cells.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Transporte/fisiologia , Replicação do DNA/fisiologia , Proteínas Nucleares/metabolismo , Proteínas Nucleares/fisiologia , Proteína BRCA1 , Proteína BRCA2 , Linhagem Celular , Quebras de DNA de Cadeia Dupla , DNA Helicases/fisiologia , Reparo do DNA , Proteínas de Ligação a DNA , Desoxirribonucleases , Endodesoxirribonucleases , Instabilidade Genômica/fisiologia , Recombinação Homóloga/genética , Humanos , Proteína Homóloga a MRE11/metabolismo , Ligação Proteica
2.
Nat Chem ; 13(6): 540-548, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33833446

RESUMO

The encoding of chemical compounds with amplifiable DNA tags facilitates the discovery of small-molecule ligands for proteins. To investigate the impact of stereo- and regiochemistry on ligand discovery, we synthesized a DNA-encoded library of 670,752 derivatives based on 2-azido-3-iodophenylpropionic acids. The library was selected against multiple proteins and yielded specific ligands. The selection fingerprints obtained for a set of protein targets of pharmaceutical relevance clearly showed the preferential enrichment of ortho-, meta- or para-regioisomers, which was experimentally verified by affinity measurements in the absence of DNA. The discovered ligands included novel selective enzyme inhibitors and binders to tumour-associated antigens, which enabled conditional chimeric antigen receptor T-cell activation and tumour targeting.


Assuntos
Sistemas de Liberação de Medicamentos , Região Variável de Imunoglobulina/farmacologia , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Animais , Linhagem Celular Tumoral , DNA/química , Descoberta de Drogas , Fluorescência , Biblioteca Gênica , Humanos , Região Variável de Imunoglobulina/química , Camundongos , Microscopia de Fluorescência , Neoplasias , Neoplasias Experimentais
3.
Adv Sci (Weinh) ; 7(22): 2001970, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33240760

RESUMO

A versatile and Lipinski-compliant DNA-encoded library (DEL), comprising 366 600 glutamic acid derivatives coupled to oligonucleotides serving as amplifiable identification barcodes is designed, constructed, and characterized. The GB-DEL library, constructed in single-stranded DNA format, allows de novo identification of specific binders against several pharmaceutically relevant proteins. Moreover, hybridization of the single-stranded DEL with a set of known protein ligands of low to medium affinity coupled to a complementary DNA strand results in self-assembled selectable chemical structures, leading to the identification of affinity-matured compounds.

4.
Cell Rep ; 32(8): 108068, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32846126

RESUMO

Using genome-wide radiogenetic profiling, we functionally dissect vulnerabilities of cancer cells to ionizing radiation (IR). We identify ERCC6L2 as a major determinant of IR response, together with classical DNA damage response genes and members of the recently identified shieldin and CTC1-STN1-TEN1 (CST) complexes. We show that ERCC6L2 contributes to non-homologous end joining (NHEJ), and it may exert this function through interactions with SFPQ. In addition to causing radiosensitivity, ERCC6L2 loss restores DNA end resection and partially rescues homologous recombination (HR) in BRCA1-deficient cells. As a consequence, ERCC6L2 deficiency confers resistance to poly (ADP-ribose) polymerase (PARP) inhibition in tumors deficient for both BRCA1 and p53. Moreover, we show that ERCC6L2 mutations are found in human tumors and correlate with a better overall survival in patients treated with radiotherapy (RT); this finding suggests that ERCC6L2 is a predictive biomarker of RT response.


Assuntos
Reparo do DNA por Junção de Extremidades/efeitos da radiação , DNA Helicases/metabolismo , Animais , Humanos , Camundongos
5.
Front Oncol ; 9: 1388, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31921645

RESUMO

DNA double-strand breaks (DSBs) are highly deleterious, with a single unrepaired DSB being sufficient to trigger cell death. Compared to healthy cells, cancer cells have a higher DSB burden due to oncogene-induced replication stress and acquired defects in DNA damage response (DDR) mechanisms. Consequently, hyperproliferating cancer cells rely on efficient DSB repair for their survival. Moreover, augmented DSB repair capacity is a major cause of radio- and chemoresistance and, ultimately, cancer recurrence. Although inherited DDR defects can predispose individuals to develop certain cancers, the very same vulnerability may be therapeutically exploited to preferentially kill tumor cells. A paradigm for DNA repair targeted therapy has emerged in cancers that exhibit mutations in BRCA1 or BRCA2 tumor suppressor genes, conferring a strong defect in homologous recombination, a major and error-free DSB repair pathway. Clinical validation of such approaches, commonly described as synthetic lethality (SL), has been provided by the regulatory approval of poly(ADP-ribose) polymerase 1 inhibitors (PARPi) as monotherapy for BRCA1/2-mutated breast and ovarian tumors. In this review, we will describe the different DSB repair mechanisms and discuss how their specific features could be exploited for cancer therapy. A major emphasis is put on advances in combinatorial treatment modalities and SL approaches arising from DSB repair pathway interdependencies.

6.
DNA Repair (Amst) ; 77: 96-108, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30928893

RESUMO

DNA double-strand breaks (DSBs) induced by genotoxic agents can cause cell death or contribute to chromosomal instability, a major driving force of cancer. By contrast, Spo11-dependent DSBs formed during meiosis are aimed at generating genetic diversity. In eukaryotes, CtIP and the Mre11 nuclease complex are essential for accurate processing and repair of both unscheduled and programmed DSBs by homologous recombination (HR). Here, we applied bioinformatics and genetic analysis to identify Paramecium tetraurelia CtIP (PtCtIP), the smallest known Sae2/Ctp1/CtIP ortholog, as a key factor for the completion of meiosis and the recovery of viable sexual progeny. Using in vitro assays, we find that purified recombinant PtCtIP preferentially binds to double-stranded DNA substrates but does not contain intrinsic nuclease activity. Moreover, mutation of the evolutionarily conserved C-terminal 'RHR' motif abrogates DNA binding of PtCtIP but not its ability to functionally interact with Mre11. Translating our findings into mammalian cells, we provide evidence that disruption of the 'RHR' motif abrogates accumulation of human CtIP at sites of DSBs. Consequently, cells expressing the DNA binding mutant CtIPR837A/R839A are defective in DSB resection and HR. Collectively, our work highlights minimal structural requirements for CtIP protein family members to facilitate the processing of DSBs, thereby maintaining genome stability as well as enabling sexual reproduction.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Paramecium tetraurellia/genética , Paramecium tetraurellia/fisiologia , Proteínas de Protozoários/metabolismo , Homologia de Sequência de Aminoácidos , Motivos de Aminoácidos , Sequência de Aminoácidos , Sequência Conservada , DNA de Protozoário/metabolismo , Meiose/genética , Paramecium tetraurellia/metabolismo , Proteínas de Protozoários/química , Reprodução/genética
7.
Mol Cancer Ther ; 17(7): 1392-1404, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29654063

RESUMO

Under conditions of genotoxic stress, cancer cells strongly rely on efficient DNA repair to survive and proliferate. The human BRCA2 tumor suppressor protein is indispensable for the repair of DNA double-strand breaks by homologous recombination (HR) by virtue of its ability to promote RAD51 loading onto single-stranded DNA. Therefore, blocking the interaction between BRCA2 and RAD51 could significantly improve the efficacy of conventional anticancer therapies. However, targeting protein-protein interaction (PPI) interfaces has proven challenging because flat and large PPI surfaces generally do not support binding of small-molecule inhibitors. In contrast, peptides are more potent for targeting PPIs but are otherwise difficult to deliver into cells. Here, we report that a synthetic 16-mer peptide derived from the BRC4 repeat motif of BRCA2 is capable of blocking RAD51 binding to BRCA2. Efficient noncytotoxic cellular uptake of a nona-arginine (R9)-conjugated version of the BRC4 peptide interferes with DNA damage-induced RAD51 foci formation and HR. Moreover, transduction of the BRC4 peptide impairs replication fork-protective function of BRCA2 and triggers MRE11-dependent degradation of nascent DNA in response to DNA replication stress. Finally, the BRC4 cell-penetrating peptide (CPP) confers selective hypersensitivity to PARP inhibition in cancer cells but spares noncancerous cells. Taken together, our data highlight an innovative approach to develop novel peptide-based DNA repair inhibitors and establish BRCA2-derived CPPs as promising anticancer agents. Mol Cancer Ther; 17(7); 1392-404. ©2018 AACR.


Assuntos
Proteína BRCA2/metabolismo , Peptídeos Penetradores de Células/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Rad51 Recombinase/metabolismo , Sequência de Aminoácidos , Proteína BRCA2/química , Linhagem Celular Tumoral , Peptídeos Penetradores de Células/química , Replicação do DNA/efeitos dos fármacos , Recombinação Homóloga/efeitos dos fármacos , Humanos , Proteína Homóloga a MRE11/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/química , Ligação Proteica/efeitos dos fármacos , Proteólise , Rad51 Recombinase/antagonistas & inibidores
8.
Nat Commun ; 7: 12628, 2016 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-27561354

RESUMO

Human CtIP is a decisive factor in DNA double-strand break repair pathway choice by enabling DNA-end resection, the first step that differentiates homologous recombination (HR) from non-homologous end-joining (NHEJ). To coordinate appropriate and timely execution of DNA-end resection, CtIP function is tightly controlled by multiple protein-protein interactions and post-translational modifications. Here, we identify the Cullin3 E3 ligase substrate adaptor Kelch-like protein 15 (KLHL15) as a new interaction partner of CtIP and show that KLHL15 promotes CtIP protein turnover via the ubiquitin-proteasome pathway. A tripeptide motif (FRY) conserved across vertebrate CtIP proteins is essential for KLHL15-binding; its mutation blocks KLHL15-dependent CtIP ubiquitination and degradation. Consequently, DNA-end resection is strongly attenuated in cells overexpressing KLHL15 but amplified in cells either expressing a CtIP-FRY mutant or lacking KLHL15, thus impacting the balance between HR and NHEJ. Collectively, our findings underline the key importance and high complexity of CtIP modulation for genome integrity.


Assuntos
Proteínas de Transporte/metabolismo , Reparo do DNA por Junção de Extremidades , Recombinação Homóloga/genética , Proteínas dos Microfilamentos/metabolismo , Proteínas Nucleares/metabolismo , Ubiquitinação/genética , Proteínas de Transporte/genética , Linhagem Celular , Proteínas Culina/metabolismo , Quebras de DNA de Cadeia Dupla , Endodesoxirribonucleases , Humanos , Mutação , Proteínas Nucleares/genética , Domínios e Motivos de Interação entre Proteínas/genética , Proteólise
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