RESUMO
The Werner Syndrome helicase, WRN, is a promising therapeutic target in cancers with microsatellite instability (MSI). Long-term MSI leads to the expansion of TA nucleotide repeats proposed to form cruciform DNA structures, which in turn cause DNA breaks and cell lethality upon WRN downregulation. Here we employed biochemical assays to show that WRN helicase can efficiently and directly unfold cruciform structures, thereby preventing their cleavage by the SLX1-SLX4 structure-specific endonuclease. TA repeats are particularly prone to form cruciform structures, explaining why these DNA sequences are preferentially broken in MSI cells upon WRN downregulation. We further demonstrate that the activity of the DNA mismatch repair (MMR) complexes MutSα (MSH2-MSH6), MutSß (MSH2-MSH3), and MutLα (MLH1-PMS2) similarly decreases the level of DNA cruciforms, although the mechanism is different from that employed by WRN. When combined, WRN and MutLα exhibited higher than additive effects in in vitro cruciform processing, suggesting that WRN and the MMR proteins may cooperate. Our data explain how WRN and MMR defects cause genome instability in MSI cells with expanded TA repeats, and provide a mechanistic basis for their recently discovered synthetic-lethal interaction with promising applications in precision cancer therapy.
Assuntos
Reparo de Erro de Pareamento de DNA , DNA Cruciforme , Humanos , Proteína 2 Homóloga a MutS/genética , Proteína 2 Homóloga a MutS/metabolismo , Instabilidade de Microssatélites , Helicase da Síndrome de Werner/genética , Helicase da Síndrome de Werner/metabolismo , Proteína 1 Homóloga a MutL/genéticaRESUMO
Defects in DNA repair frequently lead to neurodevelopmental and neurodegenerative diseases, underscoring the particular importance of DNA repair in long-lived post-mitotic neurons1,2. The cellular genome is subjected to a constant barrage of endogenous DNA damage, but surprisingly little is known about the identity of the lesion(s) that accumulate in neurons and whether they accrue throughout the genome or at specific loci. Here we show that post-mitotic neurons accumulate unexpectedly high levels of DNA single-strand breaks (SSBs) at specific sites within the genome. Genome-wide mapping reveals that SSBs are located within enhancers at or near CpG dinucleotides and sites of DNA demethylation. These SSBs are repaired by PARP1 and XRCC1-dependent mechanisms. Notably, deficiencies in XRCC1-dependent short-patch repair increase DNA repair synthesis at neuronal enhancers, whereas defects in long-patch repair reduce synthesis. The high levels of SSB repair in neuronal enhancers are therefore likely to be sustained by both short-patch and long-patch processes. These data provide the first evidence of site- and cell-type-specific SSB repair, revealing unexpected levels of localized and continuous DNA breakage in neurons. In addition, they suggest an explanation for the neurodegenerative phenotypes that occur in patients with defective SSB repair.
Assuntos
Quebras de DNA de Cadeia Simples , Reparo do DNA , Elementos Facilitadores Genéticos/genética , Neurônios/metabolismo , 5-Metilcitosina/metabolismo , Linhagem Celular , DNA/biossíntese , Replicação do DNA , Humanos , Masculino , Metilação , Poli(ADP-Ribose) Polimerases/metabolismo , Análise de Sequência de DNARESUMO
The RecQ DNA helicase WRN is a synthetic lethal target for cancer cells with microsatellite instability (MSI), a form of genetic hypermutability that arises from impaired mismatch repair1-4. Depletion of WRN induces widespread DNA double-strand breaks in MSI cells, leading to cell cycle arrest and/or apoptosis. However, the mechanism by which WRN protects MSI-associated cancers from double-strand breaks remains unclear. Here we show that TA-dinucleotide repeats are highly unstable in MSI cells and undergo large-scale expansions, distinct from previously described insertion or deletion mutations of a few nucleotides5. Expanded TA repeats form non-B DNA secondary structures that stall replication forks, activate the ATR checkpoint kinase, and require unwinding by the WRN helicase. In the absence of WRN, the expanded TA-dinucleotide repeats are susceptible to cleavage by the MUS81 nuclease, leading to massive chromosome shattering. These findings identify a distinct biomarker that underlies the synthetic lethal dependence on WRN, and support the development of therapeutic agents that target WRN for MSI-associated cancers.
Assuntos
Quebras de DNA de Cadeia Dupla , Expansão das Repetições de DNA/genética , Repetições de Dinucleotídeos/genética , Neoplasias/genética , Helicase da Síndrome de Werner/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular Tumoral , Cromossomos Humanos/genética , Cromossomos Humanos/metabolismo , Cromotripsia , Clivagem do DNA , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Endonucleases/metabolismo , Instabilidade Genômica , Humanos , Recombinases/metabolismoRESUMO
Radiotherapy and chemotherapy are effective treatment methods for many types of cancer, but resistance is common. Recent findings indicate that antiviral type I interferon (IFN) signaling is induced by these treatments. However, the underlying mechanisms still need to be elucidated. Expression of a set of IFN-stimulated genes comprises an IFN-related DNA damage resistance signature (IRDS), which correlates strongly with resistance to radiotherapy and chemotherapy across different tumors. Classically, during viral infection, the presence of foreign DNA in the cytoplasm of host cells can initiate type I IFN signaling. Here, we demonstrate that DNA-damaging modalities used during cancer therapy lead to the release of ssDNA fragments from the cell nucleus into the cytosol, engaging this innate immune response. We found that the factors that control DNA end resection during double-strand break repair, including the Bloom syndrome (BLM) helicase and exonuclease 1 (EXO1), play a major role in generating these DNA fragments and that the cytoplasmic 3'-5' exonuclease Trex1 is required for their degradation. Analysis of mRNA expression profiles in breast tumors demonstrates that those with lower Trex1 and higher BLM and EXO1 expression levels are associated with poor prognosis. Targeting BLM and EXO1 could therefore represent a novel approach for circumventing the IRDS produced in response to cancer therapeutics.
Assuntos
Dano ao DNA , Exodesoxirribonucleases/metabolismo , Imunidade Inata/genética , Interferons/metabolismo , Fosfoproteínas/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Neoplasias da Mama/terapia , Linhagem Celular Tumoral , Citoplasma/enzimologia , Citoplasma/imunologia , Citoplasma/metabolismo , Dano ao DNA/efeitos dos fármacos , DNA de Cadeia Simples/imunologia , DNA de Cadeia Simples/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Camundongos , Mutagênicos/uso terapêutico , Mutagênicos/toxicidade , Tolerância a Radiação/imunologia , Radiação Ionizante , Espécies Reativas de Oxigênio , Transdução de SinaisRESUMO
The SARS-CoV-2 coronavirus is the causal agent of the current global pandemic. SARS-CoV-2 belongs to an order, Nidovirales, with very large RNA genomes. It is proposed that the fidelity of coronavirus (CoV) genome replication is aided by an RNA nuclease complex, comprising the non-structural proteins 14 and 10 (nsp14-nsp10), an attractive target for antiviral inhibition. Our results validate reports that the SARS-CoV-2 nsp14-nsp10 complex has RNase activity. Detailed functional characterization reveals nsp14-nsp10 is a versatile nuclease capable of digesting a wide variety of RNA structures, including those with a blocked 3'-terminus. Consistent with a role in maintaining viral genome integrity during replication, we find that nsp14-nsp10 activity is enhanced by the viral RNA-dependent RNA polymerase complex (RdRp) consisting of nsp12-nsp7-nsp8 (nsp12-7-8) and demonstrate that this stimulation is mediated by nsp8. We propose that the role of nsp14-nsp10 in maintaining replication fidelity goes beyond classical proofreading by purging the nascent replicating RNA strand of a range of potentially replication-terminating aberrations. Using our developed assays, we identify drug and drug-like molecules that inhibit nsp14-nsp10, including the known SARS-CoV-2 major protease (Mpro) inhibitor ebselen and the HIV integrase inhibitor raltegravir, revealing the potential for multifunctional inhibitors in COVID-19 treatment.
Assuntos
Antivirais/farmacologia , Avaliação Pré-Clínica de Medicamentos , Exorribonucleases/metabolismo , Genoma Viral/genética , Instabilidade Genômica , SARS-CoV-2/enzimologia , SARS-CoV-2/genética , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , RNA-Polimerase RNA-Dependente de Coronavírus/metabolismo , Exorribonucleases/antagonistas & inibidores , Genoma Viral/efeitos dos fármacos , Instabilidade Genômica/efeitos dos fármacos , Instabilidade Genômica/genética , Inibidores de Integrase de HIV/farmacologia , Isoindóis/farmacologia , Complexos Multienzimáticos/antagonistas & inibidores , Complexos Multienzimáticos/metabolismo , Compostos Organosselênicos/farmacologia , RNA Viral/biossíntese , RNA Viral/genética , Raltegravir Potássico/farmacologia , SARS-CoV-2/efeitos dos fármacos , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas Virais Reguladoras e Acessórias/antagonistas & inibidores , Replicação Viral/efeitos dos fármacos , Replicação Viral/genéticaRESUMO
The SNM1 nucleases which help maintain genome integrity are members of the metallo-ß-lactamase (MBL) structural superfamily. Their conserved MBL-ß-CASP-fold SNM1 core provides a molecular scaffold forming an active site which coordinates the metal ions required for catalysis. The features that determine SNM1 endo- versus exonuclease activity, and which control substrate selectivity and binding are poorly understood. We describe a structure of SNM1B/Apollo with two nucleotides bound to its active site, resembling the product state of its exonuclease reaction. The structure enables definition of key SNM1B residues that form contacts with DNA and identifies a 5' phosphate binding pocket, which we demonstrate is important in catalysis and which has a key role in determining endo- versus exonucleolytic activity across the SNM1 family. We probed the capacity of SNM1B to digest past sites of common endogenous DNA lesions and find that base modifications planar to the nucleobase can be accommodated due to the open architecture of the active site, but lesions axial to the plane of the nucleobase are not well tolerated due to constriction around the altered base. We propose that SNM1B/Apollo might employ its activity to help remove common oxidative lesions from telomeres.
Assuntos
Endonucleases/química , Exodesoxirribonucleases/química , Exonucleases/química , beta-Lactamases/genética , Sítios de Ligação/genética , Catálise , Domínio Catalítico/genética , Proteínas de Ligação a DNA , Endonucleases/genética , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/ultraestrutura , Exonucleases/genética , Humanos , Metais , Fosfatos/química , beta-Lactamases/químicaRESUMO
Artemis (SNM1C/DCLRE1C) is an endonuclease that plays a key role in development of B- and T-lymphocytes and in dsDNA break repair by non-homologous end-joining (NHEJ). Artemis is phosphorylated by DNA-PKcs and acts to open DNA hairpin intermediates generated during V(D)J and class-switch recombination. Artemis deficiency leads to congenital radiosensitive severe acquired immune deficiency (RS-SCID). Artemis belongs to a superfamily of nucleases containing metallo-ß-lactamase (MBL) and ß-CASP (CPSF-Artemis-SNM1-Pso2) domains. We present crystal structures of the catalytic domain of wildtype and variant forms of Artemis, including one causing RS-SCID Omenn syndrome. The catalytic domain of the Artemis has similar endonuclease activity to the phosphorylated full-length protein. Our structures help explain the predominantly endonucleolytic activity of Artemis, which contrasts with the predominantly exonuclease activity of the closely related SNM1A and SNM1B MBL fold nucleases. The structures reveal a second metal binding site in its ß-CASP domain unique to Artemis, which is amenable to inhibition by compounds including ebselen. By combining our structural data with that from a recently reported Artemis structure, we were able model the interaction of Artemis with DNA substrates. The structures, including one of Artemis with the cephalosporin ceftriaxone, will help enable the rational development of selective SNM1 nuclease inhibitors.
Assuntos
Proteínas de Ciclo Celular/ultraestrutura , Proteínas de Ligação a DNA/ultraestrutura , Endonucleases/ultraestrutura , Exodesoxirribonucleases/ultraestrutura , Imunodeficiência Combinada Severa/genética , Linfócitos B/enzimologia , Domínio Catalítico/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Cristalografia por Raios X , Reparo do DNA por Junção de Extremidades/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Endonucleases/antagonistas & inibidores , Endonucleases/química , Endonucleases/genética , Inibidores Enzimáticos/química , Exodesoxirribonucleases/química , Exodesoxirribonucleases/genética , Humanos , Fosforilação/genética , Dobramento de Proteína , Imunodeficiência Combinada Severa/enzimologia , Imunodeficiência Combinada Severa/patologia , Linfócitos T/enzimologiaRESUMO
BACKGROUND: Congenital dyserythropoietic anaemia type I (CDA-I) is a hereditary anaemia caused by biallelic mutations in the widely expressed genes CDAN1 and C15orf41. Little is understood about either protein and it is unclear in which cellular pathways they participate. METHODS: Genetic analysis of a cohort of patients with CDA-I identifies novel pathogenic variants in both known causative genes. We analyse the mutation distribution and the predicted structural positioning of amino acids affected in Codanin-1, the protein encoded by CDAN1. Using western blotting, immunoprecipitation and immunofluorescence, we determine the effect of particular mutations on both proteins and interrogate protein interaction, stability and subcellular localisation. RESULTS: We identify six novel CDAN1 mutations and one novel mutation in C15orf41 and uncover evidence of further genetic heterogeneity in CDA-I. Additionally, population genetics suggests that CDA-I is more common than currently predicted. Mutations are enriched in six clusters in Codanin-1 and tend to affect buried residues. Many missense and in-frame mutations do not destabilise the entire protein. Rather C15orf41 relies on Codanin-1 for stability and both proteins, which are enriched in the nucleolus, interact to form an obligate complex in cells. CONCLUSION: Stability and interaction data suggest that C15orf41 may be the key determinant of CDA-I and offer insight into the mechanism underlying this disease. Both proteins share a common pathway likely to be present in a wide variety of cell types; however, nucleolar enrichment may provide a clue as to the erythroid specific nature of CDA-I. The surprisingly high predicted incidence of CDA-I suggests that better ascertainment would lead to improved patient care.
Assuntos
Anemia Diseritropoética Congênita/genética , Predisposição Genética para Doença , Glicoproteínas/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Anemia Diseritropoética Congênita/patologia , Feminino , Regulação da Expressão Gênica/genética , Testes Genéticos , Genética Populacional , Humanos , Masculino , Complexos Multiproteicos/genética , Mutação/genéticaRESUMO
During replication-coupled DNA interstrand crosslink (ICL) repair, the XPF-ERCC1 endonuclease is required for the incisions that release, or "unhook", ICLs, but the mechanism of ICL unhooking remains largely unknown. Incisions are triggered when the nascent leading strand of a replication fork strikes the ICL Here, we report that while purified XPF-ERCC1 incises simple ICL-containing model replication fork structures, the presence of a nascent leading strand, modelling the effects of replication arrest, inhibits this activity. Strikingly, the addition of the single-stranded DNA (ssDNA)-binding replication protein A (RPA) selectively restores XPF-ERCC1 endonuclease activity on this structure. The 5'-3' exonuclease SNM1A can load from the XPF-ERCC1-RPA-induced incisions and digest past the crosslink to quantitatively complete the unhooking reaction. We postulate that these collaborative activities of XPF-ERCC1, RPA and SNM1A might explain how ICL unhooking is achieved in vivo.
Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Exodesoxirribonucleases/metabolismo , Proteína de Replicação A/metabolismo , Proteínas de Ciclo Celular , Humanos , Modelos BiológicosRESUMO
The CRISPR system is widely used in genome editing for biomedical research. Here, using either dual paired Cas9D10A nickases or paired Cas9 nuclease we characterize unintended larger deletions at on-target sites that frequently evade common genotyping practices. We found that unintended larger deletions are prevalent at multiple distinct loci on different chromosomes, in cultured cells and mouse embryos alike. We observed a high frequency of microhomologies at larger deletion breakpoint junctions, suggesting the involvement of microhomology-mediated end joining in their generation. In populations of edited cells, the distribution of larger deletion sizes is dependent on proximity to sgRNAs and cannot be predicted by microhomology sequences alone.
Assuntos
Sistemas CRISPR-Cas , Deleção Cromossômica , Cromossomos de Mamíferos/genética , Edição de Genes/métodos , Deleção de Sequência , Animais , Linhagem Celular , Pontos de Quebra do Cromossomo , Cromossomos de Mamíferos/metabolismo , Reparo do DNA por Junção de Extremidades , Desoxirribonuclease I/genética , Desoxirribonuclease I/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Camundongos , Modelos Genéticos , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismoRESUMO
The αßßα metallo ß-lactamase (MBL) fold (MBLf) was first observed in bacterial enzymes that catalyze the hydrolysis of almost all ß-lactam antibiotics, but is now known to be widely distributed. The MBL core protein fold is present in human enzymes with diverse biological roles, including cell detoxification pathways and enabling resistance to clinically important anticancer medicines. Human (h)MBLf enzymes can bind metals, including zinc and iron ions, and catalyze a range of chemically interesting reactions, including both redox (e.g., ETHE1) and hydrolytic processes (e.g., Glyoxalase II, SNM1 nucleases, and CPSF73). With a view to promoting basic research on MBLf enzymes and their medicinal targeting, here we summarize current knowledge of the mechanisms and roles of these important molecules.
Assuntos
Enzimas Reparadoras do DNA/química , Proteínas Mitocondriais/química , Proteínas Musculares/química , Proteínas Nucleares/química , Proteínas de Transporte Nucleocitoplasmático/química , Tioléster Hidrolases/química , Zinco/química , beta-Lactamases/química , Arabidopsis/enzimologia , Arabidopsis/genética , Bactérias/enzimologia , Bactérias/genética , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Exodesoxirribonucleases , Expressão Gênica , Humanos , Hidrólise , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Tioléster Hidrolases/genética , Tioléster Hidrolases/metabolismo , Zinco/metabolismo , beta-Lactamases/genética , beta-Lactamases/metabolismo , beta-Lactamas/química , beta-Lactamas/metabolismoRESUMO
Nine modified nucleosides, incorporating zinc-binding pharmacophores, have been synthesised and evaluated as inhibitors of the DNA repair nuclease SNM1A. The series included oxyamides, hydroxamic acids, hydroxamates, a hydrazide, a squarate ester and a squaramide. A hydroxamic acid-derived nucleoside inhibited the enzyme, offering a novel approach for potential therapeutic development through the use of rationally designed nucleoside derived inhibitors.
Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Exodesoxirribonucleases/antagonistas & inibidores , Ácidos Hidroxâmicos/farmacologia , Proteínas de Ciclo Celular/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Exodesoxirribonucleases/metabolismo , Humanos , Ácidos Hidroxâmicos/síntese química , Ácidos Hidroxâmicos/química , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
One of the major DNA interstrand cross-link (ICL) repair pathways in mammalian cells is coupled to replication, but the mechanistic roles of the critical factors involved remain largely elusive. Here, we show that purified human SNM1A (hSNM1A), which exhibits a 5'-3' exonuclease activity, can load from a single DNA nick and digest past an ICL on its substrate strand. hSNM1A-depleted cells are ICL-sensitive and accumulate replication-associated DNA double-strand breaks (DSBs), akin to ERCC1-depleted cells. These DSBs are Mus81-induced, indicating that replication fork cleavage by Mus81 results from the failure of the hSNM1A- and XPF-ERCC1-dependent ICL repair pathway. Our results reveal how collaboration between hSNM1A and XPF-ERCC1 is necessary to initiate ICL repair in replicating human cells.
Assuntos
Enzimas Reparadoras do DNA/metabolismo , Reparo do DNA/genética , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Endonucleases/metabolismo , Proteínas Nucleares/metabolismo , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Quebras de DNA de Cadeia Simples , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Exodesoxirribonucleases , Células HeLa , Humanos , Proteínas Nucleares/genéticaRESUMO
The human SNM1A and SNM1B/Apollo proteins are members of an extended family of eukaryotic nuclease containing a motif related to the prokaryotic metallo-ß-lactamase (MBL) fold. SNM1A is a key exonuclease during replication-dependent and transcription-coupled interstrand crosslink repair, while SNM1B/Apollo is required for maintaining telomeric overhangs. Here, we report the crystal structures of SNM1A and SNM1B at 2.16 Å. While both proteins contain a typical MBL-ß-CASP domain, a region of positive charge surrounds the active site of SNM1A, which is absent in SNM1B and explains the greater apparent processivity of SNM1A. The structures of both proteins also reveal a putative, wide DNA-binding groove. Extensive mutagenesis of this groove, coupled with detailed biochemical analysis, identified residues that did not impact on SNM1A catalytic activity, but drastically reduced its processivity. Moreover, we identified a key role for this groove for efficient digestion past DNA interstrand crosslinks, facilitating the key DNA repair reaction catalysed by SNM1A. Together, the architecture and dimensions of this groove, coupled to the surrounding region of high positive charge, explain the remarkable ability of SNM1A to accommodate and efficiently digest highly distorted DNA substrates, such as those containing DNA lesions.
Assuntos
Enzimas Reparadoras do DNA/química , DNA/metabolismo , Exodesoxirribonucleases/química , Proteínas Nucleares/química , Domínio Catalítico , Proteínas de Ciclo Celular , Dano ao DNA , Enzimas Reparadoras do DNA/metabolismo , Exodesoxirribonucleases/metabolismo , Humanos , Modelos Moleculares , Proteínas Nucleares/metabolismo , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
Cockayne syndrome (CS) is a premature aging disorder characterized by photosensitivity, impaired development and multisystem progressive degeneration, and consists of two strict complementation groups, A and B. Using a yeast two-hybrid approach, we identified the 5'-3' exonuclease SNM1A as one of four strong interacting partners of CSB. This direct interaction was confirmed using purified recombinant proteins-with CSB able to modulate the exonuclease activity of SNM1A on oligonucleotide substrates in vitro-and the two proteins were shown to exist in a common complex in human cell extracts. CSB and SNM1A were also found, using fluorescently tagged proteins in combination with confocal microscopy and laser microirradiation, to be recruited to localized trioxsalen-induced ICL damage in human cells, with accumulation being suppressed by transcription inhibition. Moreover, SNM1A recruitment was significantly reduced in CSB-deficient cells, suggesting coordination between the two proteins in vivo. CSB-deficient neural cells exhibited increased sensitivity to DNA crosslinking agents, particularly, in a non-cycling, differentiated state, as well as delayed ICL processing as revealed by a modified Comet assay and γ-H2AX foci persistence. The results indicate that CSB coordinates the resolution of ICLs, possibly in a transcription-associated repair mechanism involving SNM1A, and that defects in the process could contribute to the post-mitotic degenerative pathologies associated with CS.
Assuntos
DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular , Reagentes de Ligações Cruzadas , DNA/metabolismo , Dano ao DNA , Exodesoxirribonucleases , Exonucleases/metabolismo , Células HeLa , Humanos , Proteínas de Ligação a Poli-ADP-RiboseRESUMO
ß-Lactams are the most successful antibacterials, but their effectiveness is threatened by resistance, most importantly by production of serine- and metallo-ß-lactamases (MBLs). MBLs are of increasing concern because they catalyze the hydrolysis of almost all ß-lactam antibiotics, including recent-generation carbapenems. Clinically useful serine-ß-lactamase inhibitors have been developed, but such inhibitors are not available for MBLs. l-Captopril, which is used to treat hypertension via angiotensin-converting enzyme inhibition, has been reported to inhibit MBLs by chelating the active site zinc ions via its thiol(ate). We report systematic studies on B1 MBL inhibition by all four captopril stereoisomers. High-resolution crystal structures of three MBLs (IMP-1, BcII, and VIM-2) in complex with either the l- or d-captopril stereoisomer reveal correlations between the binding mode and inhibition potency. The results will be useful in the design of MBL inhibitors with the breadth of selectivity required for clinical application against carbapenem-resistant Enterobacteriaceae and other organisms causing MBL-mediated resistant infections.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Captopril/farmacologia , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/química , Inibidores da Enzima Conversora de Angiotensina/química , Antibacterianos/farmacologia , Captopril/química , Carbapenêmicos/farmacologia , Clonagem Molecular , Cristalografia por Raios X , Reposicionamento de Medicamentos , Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/enzimologia , Enterobacteriaceae/genética , Expressão Gênica , Hidrólise , Cinética , Testes de Sensibilidade Microbiana , Modelos Moleculares , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Estereoisomerismo , Relação Estrutura-Atividade , Resistência beta-Lactâmica/efeitos dos fármacos , Resistência beta-Lactâmica/genética , Inibidores de beta-Lactamases/química , beta-Lactamases/genética , beta-Lactamases/metabolismoRESUMO
Fanconi anemia (FA) is a devastating genetic disease, associated with genomic instability and defects in DNA interstrand cross-link (ICL) repair. The FA repair pathway is not thought to be conserved in budding yeast, and although the yeast Mph1 helicase is a putative homolog of human FANCM, yeast cells disrupted for MPH1 are not sensitive to ICLs. Here, we reveal a key role for Mph1 in ICL repair when the Pso2 exonuclease is inactivated. We find that the yeast FANCM ortholog Mph1 physically and functionally interacts with Mgm101, a protein previously implicated in mitochondrial DNA repair, and the MutSα mismatch repair factor (Msh2-Msh6). Co-disruption of MPH1, MGM101, MSH6, or MSH2 with PSO2 produces a lesion-specific increase in ICL sensitivity, the elevation of ICL-induced chromosomal rearrangements, and persistence of ICL-associated DNA double-strand breaks. We find that Mph1-Mgm101-MutSα directs the ICL-induced recruitment of Exo1 to chromatin, and we propose that Exo1 is an alternative 5'-3' exonuclease utilised for ICL repair in the absence of Pso2. Moreover, ICL-induced Rad51 chromatin loading is delayed when both Pso2 and components of the Mph1-Mgm101-MutSα and Exo1 pathway are inactivated, demonstrating that the homologous recombination stages of ICL repair are inhibited. Finally, the FANCJ- and FANCP-related factors Chl1 and Slx4, respectively, are also components of the genetic pathway controlled by Mph1-Mgm101-MutSα. Together this suggests that a prototypical FA-related ICL repair pathway operates in budding yeast, which acts redundantly with the pathway controlled by Pso2, and is required for the targeting of Exo1 to chromatin to execute ICL repair.
Assuntos
RNA Helicases DEAD-box/genética , Reparo do DNA , Endodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , RNA Helicases DEAD-box/deficiência , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/deficiência , Endodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/genética , Anemia de Fanconi/genética , Humanos , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Modelos Biológicos , Proteína 2 Homóloga a MutS/genética , Proteína 2 Homóloga a MutS/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais/genéticaRESUMO
As the options for systemic treatment of malignant melanoma (MM) increase, the need to develop biomarkers to identify patients who might benefit from cytotoxic chemotherapy becomes more apparent. In preclinical models, oxaliplatin has activity in cisplatin-resistant cells. In this study, we have shown that oxaliplatin forms interstrand crosslinks (ICLs) in cellular DNA and that loss of the heterodimeric structure-specific endonuclease XPF-ERCC1 causes hypersensitivity to oxaliplatin in mammalian cells. XPF deficiency resulted in late S-phase arrest and persistence of double-strand breaks following oxaliplatin treatment. In a panel of 12 MM cell lines, oxaliplatin sensitivity correlated with XPF and ERCC1 protein levels. The knockdown of ERCC1 and XPF protein levels by RNA interference increased sensitivity of cancer cells to oxaliplatin; overexpression of exogenous ERCC1 significantly decreased drug sensitivity. Following immunohistochemical optimization, XPF protein levels were quantified in MM tissue samples from 183 patients, showing variation in expression and no correlation with prognosis. In 57 patients with MM treated with cisplatin or carboplatin, XPF protein levels did not predict the likelihood of clinical response. We propose that oxaliplatin should not be discarded as a potential treatment for MM on the basis of the limited activity of cisplatin in unselected patients. Moreover, we show that XPF-ERCC1 protein levels are a key determinant of the sensitivity of melanoma cells to oxaliplatin in vitro. Immunohistochemical detection of XPF appears suitable for development as a tissue biomarker for potentially selecting patients for oxaliplatin treatment in a prospective clinical trial.
Assuntos
Antineoplásicos/farmacologia , Biomarcadores Tumorais/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Melanoma/tratamento farmacológico , Compostos Organoplatínicos/farmacologia , Fatores de Transcrição/metabolismo , Estudos de Coortes , Dano ao DNA/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Humanos , Técnicas Imunoenzimáticas , Melanoma/metabolismo , Melanoma/patologia , Pessoa de Meia-Idade , Oxaliplatina , Seleção de Pacientes , Fase S/efeitos dos fármacos , Neoplasias Cutâneas , Análise Serial de Tecidos , Células Tumorais Cultivadas , Melanoma Maligno CutâneoRESUMO
DNA double-strand breaks (DSBs), such as those produced by radiation and radiomimetics, are amongst the most toxic forms of cellular damage, in part because they involve extensive oxidative modifications at the break termini. Prior to completion of DSB repair, the chemically modified termini must be removed. Various DNA processing enzymes have been implicated in the processing of these dirty ends, but molecular knowledge of this process is limited. Here, we demonstrate a role for the metallo-ß-lactamase fold 5'-3' exonuclease SNM1A in this vital process. Cells disrupted for SNM1A manifest increased sensitivity to radiation and radiomimetic agents and show defects in DSB damage repair. SNM1A is recruited and is retained at the sites of DSB damage via the concerted action of its three highly conserved PBZ, PIP box and UBZ interaction domains, which mediate interactions with poly-ADP-ribose chains, PCNA and the ubiquitinated form of PCNA, respectively. SNM1A can resect DNA containing oxidative lesions induced by radiation damage at break termini. The combined results reveal a crucial role for SNM1A to digest chemically modified DNA during the repair of DSBs and imply that the catalytic domain of SNM1A is an attractive target for potentiation of radiotherapy.