RESUMO
Intrinsic and acquired drug resistance and induction of secondary malignancies limit successful chemotherapy. Because mutagenic translesion synthesis (TLS) contributes to chemoresistance as well as treatment-induced mutations, targeting TLS is an attractive avenue for improving chemotherapeutics. However, development of small molecules with high specificity and in vivo efficacy for mutagenic TLS has been challenging. Here, we report the discovery of a small-molecule inhibitor, JH-RE-06, that disrupts mutagenic TLS by preventing recruitment of mutagenic POL ζ. Remarkably, JH-RE-06 targets a nearly featureless surface of REV1 that interacts with the REV7 subunit of POL ζ. Binding of JH-RE-06 induces REV1 dimerization, which blocks the REV1-REV7 interaction and POL ζ recruitment. JH-RE-06 inhibits mutagenic TLS and enhances cisplatin-induced toxicity in cultured human and mouse cell lines. Co-administration of JH-RE-06 with cisplatin suppresses the growth of xenograft human melanomas in mice, establishing a framework for developing TLS inhibitors as a novel class of chemotherapy adjuvants.
Assuntos
Antineoplásicos/uso terapêutico , Cisplatino/uso terapêutico , Mutagênese/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Quinolinas/uso terapêutico , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/efeitos adversos , Cisplatino/farmacologia , Dano ao DNA/efeitos dos fármacos , DNA Polimerase Dirigida por DNA , Feminino , Técnicas de Silenciamento de Genes , Humanos , Proteínas Mad2/metabolismo , Camundongos , Camundongos Nus , Camundongos Transgênicos , Neoplasias/metabolismo , Neoplasias/patologia , Nucleotidiltransferases/antagonistas & inibidores , Nucleotidiltransferases/química , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Quinolinas/química , Quinolinas/farmacologia , Transfecção , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In this issue of Molecular Cell, Rotheneder et al.1 elucidate the eukaroytic Mre11-Rad50-Nbs1 (MRN) complex quaternary architecture, which together with cryo-EM structures of bacterial Mre11-Rad50-DNA complexes,2 resolves the basis for MRN assembly and its broad nuclease specificity regulating DNA double-strand break repair.
Assuntos
Proteínas de Ciclo Celular , Enzimas Reparadoras do DNA , Proteína Homóloga a MRE11/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Reparo do DNA , DNA/genética , Hidrolases Anidrido Ácido/genéticaRESUMO
The APEX2 gene encodes APE2, a nuclease related to APE1, the apurinic/apyrimidinic endonuclease acting in base excision repair. Loss of APE2 is lethal in cells with mutated BRCA1 or BRCA2, making APE2 a prime target for homologous recombination-defective cancers. However, because the function of APE2 in DNA repair is poorly understood, it is unclear why BRCA-deficient cells require APE2 for viability. Here we present the genetic interaction profiles of APE2, APE1, and TDP1 deficiency coupled to biochemical and structural dissection of APE2. We conclude that the main role of APE2 is to reverse blocked 3' DNA ends, problematic lesions that preclude DNA synthesis. Our work also suggests that TOP1 processing of genomic ribonucleotides is the main source of 3'-blocking lesions relevant to APEX2-BRCA1/2 synthetic lethality. The exquisite sensitivity of BRCA-deficient cells to 3' blocks indicates that they represent a tractable vulnerability in homologous recombination-deficient tumor cells.
Assuntos
Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Endonucleases/metabolismo , Enzimas Multifuncionais/metabolismo , Proteína BRCA1/genética , Proteína BRCA2/genética , Linhagem Celular , DNA/metabolismo , Dano ao DNA , Reparo do DNA/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Endonucleases/genética , Genes BRCA1/fisiologia , Humanos , Enzimas Multifuncionais/genética , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismoRESUMO
The homodimeric PolG2 accessory subunit of the mitochondrial DNA polymerase gamma (Pol γ) enhances DNA binding and processive DNA synthesis by the PolG catalytic subunit. PolG2 also directly binds DNA, although the underlying molecular basis and functional significance are unknown. Here, data from Atomic Force Microscopy (AFM) and X-ray structures of PolG2-DNA complexes define dimeric and hexameric PolG2 DNA binding modes. Targeted disruption of PolG2 DNA-binding interfaces impairs processive DNA synthesis without diminishing Pol γ subunit affinities. In addition, a structure-specific DNA-binding role for PolG2 oligomers is supported by X-ray structures and AFM showing that oligomeric PolG2 localizes to DNA crossings and targets forked DNA structures resembling the mitochondrial D-loop. Overall, data indicate that PolG2 DNA binding has both PolG-dependent and -independent functions in mitochondrial DNA replication and maintenance, which provide new insight into molecular defects associated with PolG2 disruption in mitochondrial disease.
Assuntos
DNA Polimerase gama , DNA Mitocondrial , Humanos , DNA Polimerase gama/genética , DNA Polimerase gama/metabolismo , Replicação do DNA/genética , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Doenças Mitocondriais/genética , Doenças Mitocondriais/metabolismoRESUMO
The NEIL3 DNA glycosylase maintains genome integrity during replication by excising oxidized bases from single-stranded DNA (ssDNA) and unhooking interstrand cross-links (ICLs) at fork structures. In addition to its N-terminal catalytic glycosylase domain, NEIL3 contains two tandem C-terminal GRF-type zinc fingers that are absent in the other NEIL paralogs. ssDNA binding by the GRF-ZF motifs helps recruit NEIL3 to replication forks converged at an ICL, but the nature of DNA binding and the effect of the GRF-ZF domain on catalysis of base excision and ICL unhooking is unknown. Here, we show that the tandem GRF-ZFs of NEIL3 provide affinity and specificity for DNA that is greater than each individual motif alone. The crystal structure of the GRF domain shows that the tandem ZF motifs adopt a flexible head-to-tail configuration well-suited for binding to multiple ssDNA conformations. Functionally, we establish that the NEIL3 GRF domain inhibits glycosylase activity against monoadducts and ICLs. This autoinhibitory activity contrasts GRF-ZF domains of other DNA-processing enzymes, which typically use ssDNA binding to enhance catalytic activity, and suggests that the C-terminal region of NEIL3 is involved in both DNA damage recruitment and enzymatic regulation.
Assuntos
DNA de Cadeia Simples/metabolismo , N-Glicosil Hidrolases/metabolismo , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , DNA/metabolismo , Replicação do DNA , DNA de Cadeia Simples/química , Humanos , Camundongos , N-Glicosil Hidrolases/antagonistas & inibidores , N-Glicosil Hidrolases/genética , Ligação Proteica , Estrutura Terciária de Proteína , Alinhamento de Sequência , Dedos de ZincoRESUMO
The Xenopus laevis APE2 (apurinic/apyrimidinic endonuclease 2) nuclease participates in 3'-5' nucleolytic resection of oxidative DNA damage and activation of the ATR-Chk1 DNA damage response (DDR) pathway via ill-defined mechanisms. Here we report that APE2 resection activity is regulated by DNA interactions in its Zf-GRF domain, a region sharing high homology with DDR proteins Topoisomerase 3α (TOP3α) and NEIL3 (Nei-like DNA glycosylase 3), as well as transcription and RNA regulatory proteins, such as TTF2 (transcription termination factor 2), TFIIS, and RPB9. Biochemical and NMR results establish the nucleic acid-binding activity of the Zf-GRF domain. Moreover, an APE2 Zf-GRF X-ray structure and small-angle X-ray scattering analyses show that the Zf-GRF fold is typified by a crescent-shaped ssDNA binding claw that is flexibly appended to an APE2 endonuclease/exonuclease/phosphatase (EEP) catalytic core. Structure-guided Zf-GRF mutations impact APE2 DNA binding and 3'-5' exonuclease processing, and also prevent efficient APE2-dependent RPA recruitment to damaged chromatin and activation of the ATR-Chk1 DDR pathway in response to oxidative stress in Xenopus egg extracts. Collectively, our data unveil the APE2 Zf-GRF domain as a nucleic acid interaction module in the regulation of a key single-strand break resection function of APE2, and also reveal topologic similarity of the Zf-GRF to the zinc ribbon domains of TFIIS and RPB9.
Assuntos
Dano ao DNA/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Estresse Oxidativo/genética , Animais , DNA Glicosilases/metabolismo , Reparo do DNA/genética , DNA Topoisomerases Tipo I/metabolismo , Endonucleases/metabolismo , Domínios Proteicos/genética , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMO
FAAP20 is an integral component of the Fanconi anemia core complex that mediates the repair of DNA interstrand crosslinks. The ubiquitin-binding capacity of the FAAP20 UBZ is required for recruitment of the Fanconi anemia complex to interstrand DNA crosslink sites and for interaction with the translesion synthesis machinery. Although the UBZ-ubiquitin interaction is thought to be exclusively encapsulated within the ßßα module of UBZ, we show that the FAAP20-ubiquitin interaction extends beyond such a canonical zinc-finger motif. Instead, ubiquitin binding by FAAP20 is accompanied by transforming a disordered tail C-terminal to the UBZ of FAAP20 into a rigid, extended ß-loop that latches onto the complex interface of the FAAP20 UBZ and ubiquitin, with the invariant C-terminal tryptophan emanating toward I44(Ub) for enhanced binding specificity and affinity. Substitution of the C-terminal tryptophan with alanine in FAAP20 not only abolishes FAAP20-ubiquitin binding in vitro, but also causes profound cellular hypersensitivity to DNA interstrand crosslink lesions in vivo, highlighting the indispensable role of the C-terminal tail of FAAP20, beyond the compact zinc finger module, toward ubiquitin recognition and Fanconi anemia complex-mediated DNA interstrand crosslink repair.
Assuntos
Proteínas de Grupos de Complementação da Anemia de Fanconi/química , Ubiquitina/química , Reparo do DNA , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Modelos Moleculares , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Triptofano/química , Ubiquitina/metabolismoRESUMO
Since the report of "DNA untwisting" activity in 1972, â¼50 years of research has revealed seven topoisomerases in humans (TOP1, TOP1mt, TOP2α, TOP2ß, TOP3α, TOP3ß and Spo11). These conserved regulators of DNA topology catalyze controlled breakage to the DNA backbone to relieve the torsional stress that accumulates during essential DNA transactions including DNA replication, transcription, and DNA repair. Each topoisomerase-catalyzed reaction involves the formation of a topoisomerase cleavage complex (TOPcc), a covalent protein-DNA reaction intermediate formed between the DNA phosphodiester backbone and a topoisomerase catalytic tyrosine residue. A variety of perturbations to topoisomerase reaction cycles can trigger failure of the enzyme to re-ligate the broken DNA strand(s), thereby generating topoisomerase DNA-protein crosslinks (TOP-DPC). TOP-DPCs pose unique threats to genomic integrity. These complex lesions are comprised of structurally diverse protein components covalently linked to genomic DNA, which are bulky DNA adducts that can directly impact progression of the transcription and DNA replication apparatus. A variety of genome maintenance pathways have evolved to recognize and resolve TOP-DPCs. Eukaryotic cells harbor tyrosyl DNA phosphodiesterases (TDPs) that directly reverse 3'-phosphotyrosyl (TDP1) and 5'-phoshotyrosyl (TDP2) protein-DNA linkages. The broad specificity Mre11-Rad50-Nbs1 and APE2 nucleases are also critical for mitigating topoisomerase-generated DNA damage. These DNA-protein crosslink metabolizing enzymes are further enabled by proteolytic degradation, with the proteasome, Spartan, GCNA, Ddi2, and FAM111A proteases implicated thus far. Strategies to target, unfold, and degrade the protein component of TOP-DPCs have evolved as well. Here we survey mechanisms for addressing Topoisomerase 1 (TOP1) and Topoisomerase 2 (TOP2) DPCs, highlighting systems for which molecular structure information has illuminated function of these critical DNA damage response pathways.
RESUMO
Topoisomerase 1 (Top1) incises DNA containing ribonucleotides to generate complex DNA lesions that are resolved by APE2 (Apn2 in yeast). How Apn2 engages and processes this DNA damage is unclear. Here, we report X-ray crystal structures and biochemical analysis of Apn2-DNA complexes to demonstrate how Apn2 frays and cleaves 3' DNA termini via a wedging mechanism that facilitates 1-6 nucleotide endonucleolytic cleavages. APN2 deletion and DNA-wedge mutant Saccharomyces cerevisiae strains display mutator phenotypes, cell growth defects, and sensitivity to genotoxic stress in a ribonucleotide excision repair (RER)-defective background harboring a high density of Top1-incised ribonucleotides. Our data implicate a wedge-and-cut mechanism underpinning the broad-specificity Apn2 nuclease activity that mitigates mutagenic and genome instability phenotypes caused by Top1 incision at genomic ribonucleotides incorporated by DNA polymerase epsilon.
Assuntos
Proteínas de Saccharomyces cerevisiae , DNA , Dano ao DNA , DNA Polimerase II/genética , Reparo do DNA , DNA Topoisomerases Tipo I/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Ribonucleotídeos/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Cancer cells display high levels of DNA damage and replication stress, vulnerabilities that could be exploited by drugs targeting DNA repair proteins. Human CtIP promotes homology-mediated repair of DNA double-strand breaks (DSBs) and protects stalled replication forks from nucleolytic degradation, thus representing an attractive candidate for targeted cancer therapy. Here, we establish a peptide mimetic of the CtIP tetramerization motif that inhibits CtIP activity. The hydrocarbon-stapled peptide encompassing amino acid residues 18 to 28 of CtIP (SP18-28) stably binds to CtIP tetramers in vitro and facilitates their aggregation into higher-order structures. Efficient intracellular uptake of SP18-28 abrogates CtIP localization to damaged chromatin, impairs DSB repair, and triggers extensive fork degradation. Moreover, prolonged SP18-28 treatment causes hypersensitivity to DNA-damaging agents and selectively reduces the viability of BRCA1-mutated cancer cell lines. Together, our data provide a basis for the future development of CtIP-targeting compounds with the potential to treat patients with cancer.
RESUMO
Computational and experimental studies focusing on the role of conserved amino acids for folding and stability is an active and promising area of research. To date however, only a small fraction of the potential superfamilies have been investigated. To further expand our understanding we present the results of a bioinformatics analysis of the death domain superfamily. The fold consists of a six helical bundle with a Greek-key topology. Our sequence and structural studies have identified a group of conserved hydrophobic residues and corresponding long-range interactions, which we propose are important in the formation and stabilization of the hydrophobic core and native topology. Six conserved hydrophilic residues were additionally identified and may play a functional role during apoptosis. We also report the establishment of an experimental system that will facilitate studies to test the role of the conserved residues in folding and stability. Equilibrium unfolding and refolding studies of a model superfamily member, Fas-associated death domain protein indicate that the process is cooperative, two-state and reversible. Stopped-flow fluorescence studies reveal that the folding is rapid and biphasic with the majority of the hydrophobic core forming in the first phase. Site-directed mutagenesis studies indicate that conserved tryptophans 112 and 148 are important to structure, native state stability and folding. These results present the earliest conservation analysis and biophysical characterization of the Fas-associated death domain.
Assuntos
Proteína de Domínio de Morte Associada a Fas/química , Dobramento de Proteína , Triptofano/química , Sequência de Aminoácidos , Dicroísmo Circular , Biologia Computacional , Sequência Conservada , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/química , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/genética , Proteína de Domínio de Morte Associada a Fas/genética , Humanos , Interações Hidrofóbicas e Hidrofílicas , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , Estabilidade Proteica , Alinhamento de Sequência , Espectrometria de Fluorescência , Triptofano/genéticaRESUMO
CEST-NMR spectroscopy is a powerful tool for probing the conformational dynamics of macromolecules. We present a HNdec-CEST experiment that simplifies the relaxation matrix, reduces fitting parameters, and enhances signal resolution. Importantly, fitting of HNdec-CEST profiles enables robust extraction of exchange rates as well as excited-state chemical shifts and populations.