RESUMO
The XPA protein functions together with the single-stranded DNA (ssDNA) binding protein RPA as the central scaffold to ensure proper positioning of repair factors in multi-protein nucleotide excision repair (NER) machinery. We previously determined the structure of a short motif in the disordered XPA N-terminus bound to the RPA32C domain. However, a second contact between the XPA DNA-binding domain (XPA DBD) and the RPA70AB tandem ssDNA-binding domains, which is likely to influence the orientation of XPA and RPA on the damaged DNA substrate, remains poorly characterized. NMR was used to map the binding interfaces of XPA DBD and RPA70AB. Combining NMR and X-ray scattering data with comprehensive docking and refinement revealed how XPA DBD and RPA70AB orient on model NER DNA substrates. The structural model enabled design of XPA mutations that inhibit the interaction with RPA70AB. These mutations decreased activity in cell-based NER assays, demonstrating the functional importance of XPA DBD-RPA70AB interaction. Our results inform ongoing controversy about where XPA is bound within the NER bubble, provide structural insights into the molecular basis for malfunction of disease-associated XPA missense mutations, and contribute to understanding of the structure and mechanical action of the NER machinery.
Assuntos
Reparo do DNA/genética , Modelos Moleculares , Proteína de Replicação A/química , Proteína de Xeroderma Pigmentoso Grupo A/química , DNA/química , DNA/genética , Dano ao DNA/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Humanos , Espectroscopia de Ressonância Magnética , Ligação Proteica/genética , Proteína de Replicação A/genética , Proteína de Xeroderma Pigmentoso Grupo A/genéticaRESUMO
WEE1 kinase is a key regulator of the G2/M transition. The WEE1 kinase inhibitor AZD1775 (WEE1i) induces origin firing in replicating cells. We show that WEE1i induces CDK1-dependent RIF1 phosphorylation and CDK2- and CDC7-dependent activation of the replicative helicase. WEE1 suppresses CDK1 and CDK2 kinase activities to regulate the G1/S transition after the origin licensing is complete. We identify a role for WEE1 in cell cycle regulation and important effects of AZD1775, which is in clinical trials.
Assuntos
Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/fisiologia , Fase G1/efeitos dos fármacos , Proteínas Tirosina Quinases/fisiologia , Pirazóis/farmacologia , Pirimidinonas/farmacologia , Fase S/efeitos dos fármacos , Ciclo Celular/fisiologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/antagonistas & inibidores , Células HEK293 , Humanos , Fosforilação , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas de Ligação a Telômeros/metabolismoRESUMO
DNA damage-induced signaling by ATR and CHK1 inhibits DNA replication, stabilizes stalled and collapsed replication forks, and mediates the repair of multiple classes of DNA lesions. We and others have shown that ATR kinase inhibitors, three of which are currently undergoing clinical trials, induce excessive origin firing during unperturbed DNA replication, indicating that ATR kinase activity limits replication initiation in the absence of damage. However, the origins impacted and the underlying mechanism(s) have not been described. Here, we show that unperturbed DNA replication is associated with a low level of ATR and CHK1 kinase signaling and that inhibition of this signaling induces dormant origin firing at sites of ongoing replication throughout the S phase. We show that ATR and CHK1 kinase inhibitors induce RIF1 Ser2205 phosphorylation in a CDK1-dependent manner, which disrupts an interaction between RIF1 and PP1 phosphatase. Thus, ATR and CHK1 signaling suppresses CDK1 kinase activity throughout the S phase and stabilizes an interaction between RIF1 and PP1 in replicating cells. PP1 dephosphorylates key CDC7 and CDK2 kinase substrates to inhibit the assembly and activation of the replicative helicase. This mechanism limits origin firing during unperturbed DNA replication in human cells.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Quinase 1 do Ponto de Checagem/metabolismo , Replicação do DNA , Transdução de Sinais , Dano ao DNA , Fibroblastos , Células HEK293 , Humanos , Fosforilação , Proteínas de Ligação a Telômeros/metabolismoRESUMO
Xeroderma pigmentosum (XP) complementation group A (XPA) is an essential scaffolding protein in the multiprotein nucleotide excision repair (NER) machinery. The interaction of XPA with DNA is a core function of this protein; a number of mutations in the DNA-binding domain (DBD) are associated with XP disease. Although structures of the central globular domain of human XPA and data on binding of DNA substrates have been reported, the structural basis for XPA's DNA-binding activity remains unknown. X-ray crystal structures of the central globular domain of yeast XPA (Rad14) with lesion-containing DNA duplexes have provided valuable insights, but the DNA substrates used for this study do not correspond to the substrates of XPA as it functions within the NER machinery. To better understand the DNA-binding activity of human XPA in NER, we used NMR to investigate the interaction of its DBD with a range of DNA substrates. We found that XPA binds different single-stranded/double-stranded junction DNA substrates with a common surface. Comparisons of our NMR-based mapping of binding residues with the previously reported Rad14-DNA crystal structures revealed similarities and differences in substrate binding between XPA and Rad14. This includes direct evidence for DNA contacts to the residues extending C-terminally from the globular core, which are lacking in the Rad14 construct. Moreover, mutation of the XPA residue corresponding to Phe-262 in Rad14, previously reported as being critical for DNA binding, had only a moderate effect on the DNA-binding activity of XPA. The DNA-binding properties of several disease-associated mutations in the DBD were investigated. These results suggest that for XPA mutants exhibiting altered DNA-binding properties, a correlation exists between the extent of reduction in DNA-binding affinity and the severity of symptoms in XP patients.
Assuntos
Reparo do DNA , Proteína de Xeroderma Pigmentoso Grupo A/química , Substituição de Aminoácidos , Enzimas Reparadoras do DNA/química , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Humanos , Mutação de Sentido Incorreto , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia Estrutural de Proteína , Xeroderma Pigmentoso/genética , Xeroderma Pigmentoso/metabolismo , Proteína de Xeroderma Pigmentoso Grupo A/genética , Proteína de Xeroderma Pigmentoso Grupo A/metabolismoRESUMO
The S100A8/S100A9 heterodimer calprotectin (CP) functions in the host response to pathogens through a mechanism termed "nutritional immunity." CP binds Mn(2+) and Zn(2+) with high affinity and starves bacteria of these essential nutrients. Combining biophysical, structural, and microbiological analysis, we identified the molecular basis of Mn(2+) sequestration. The asymmetry of the CP heterodimer creates a single Mn(2+)-binding site from six histidine residues, which distinguishes CP from all other Mn(2+)-binding proteins. Analysis of CP mutants with altered metal-binding properties revealed that, despite both Mn(2+) and Zn(2+) being essential metals, maximal growth inhibition of multiple bacterial pathogens requires Mn(2+) sequestration. These data establish the importance of Mn(2+) sequestration in defense against infection, explain the broad-spectrum antimicrobial activity of CP relative to other S100 proteins, and clarify the impact of metal depletion on the innate immune response to infection.
Assuntos
Imunidade Inata , Complexo Antígeno L1 Leucocitário/química , Complexo Antígeno L1 Leucocitário/imunologia , Manganês/metabolismo , Substituição de Aminoácidos , Sítios de Ligação , Calgranulina A/química , Calgranulina A/genética , Calgranulina A/imunologia , Calgranulina B/química , Calgranulina B/genética , Calgranulina B/imunologia , Cristalografia por Raios X , Histidina/química , Interações Hospedeiro-Patógeno/imunologia , Humanos , Complexo Antígeno L1 Leucocitário/genética , Complexo Antígeno L1 Leucocitário/farmacologia , Modelos Moleculares , Mutagênese Sítio-Dirigida , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/imunologia , Zinco/metabolismoRESUMO
Xeroderma pigmentosum complementation group A (XPA) protein plays a critical role in the repair of DNA damage via the nucleotide excision repair (NER) pathway. XPA serves as a scaffold for NER, interacting with several other NER proteins as well as the DNA substrate. The critical importance of XPA is underscored by its association with the most severe clinical phenotypes of the genetic disorder Xeroderma pigmentosum. Many of these disease-associated mutations map to the XPA(98-219) DNA-binding domain (DBD) first reported ~20 years ago. Although multiple solution NMR structures of XPA(98-219) have been determined, the molecular basis for the interaction of this domain with DNA is only poorly characterized. In this report, we demonstrate using a fluorescence anisotropy DNA-binding assay that the previously reported XPA DBD binds DNA with substantially weaker affinity than the full-length protein. In-depth analysis of the XPA sequence suggested that the original DBD construct lacks critical basic charge and helical elements at its C-terminus. Generation and analysis of a series of C-terminal extensions beyond residue 219 yielded a stable, soluble human XPA(98-239) construct that binds to a Y-shaped ssDNA-dsDNA junction and other substrates with the same affinity as the full-length protein. Two-dimensional (15)N-(1)H NMR suggested XPA(98-239) contains the same globular core as XPA98-219 and likely undergoes a conformational change upon binding DNA. Together, our results demonstrate that the XPA DBD should be redefined and that XPA(98-239) is a suitable model to examine the DNA binding activity of human XPA.
Assuntos
DNA/metabolismo , Proteína de Xeroderma Pigmentoso Grupo A/química , Proteína de Xeroderma Pigmentoso Grupo A/metabolismo , Humanos , Modelos Moleculares , Estrutura Terciária de ProteínaRESUMO
Acinetobacter baumannii is an important nosocomial pathogen that accounts for up to 20 percent of infections in intensive care units worldwide. Furthermore, A. baumannii strains have emerged that are resistant to all available antimicrobials. These facts highlight the dire need for new therapeutic strategies to combat this growing public health threat. Given the critical role for transition metals at the pathogen-host interface, interrogating the role for these metals in A. baumannii physiology and pathogenesis could elucidate novel therapeutic strategies. Toward this end, the role for calprotectin- (CP)-mediated chelation of manganese (Mn) and zinc (Zn) in defense against A. baumannii was investigated. These experiments revealed that CP inhibits A. baumannii growth in vitro through chelation of Mn and Zn. Consistent with these in vitro data, Imaging Mass Spectrometry revealed that CP accompanies neutrophil recruitment to the lung and accumulates at foci of infection in a murine model of A. baumannii pneumonia. CP contributes to host survival and control of bacterial replication in the lung and limits dissemination to secondary sites. Using CP as a probe identified an A. baumannii Zn acquisition system that contributes to Zn uptake, enabling this organism to resist CP-mediated metal chelation, which enhances pathogenesis. Moreover, evidence is provided that Zn uptake across the outer membrane is an energy-dependent process in A. baumannii. Finally, it is shown that Zn limitation reverses carbapenem resistance in multidrug resistant A. baumannii underscoring the clinical relevance of these findings. Taken together, these data establish Zn acquisition systems as viable therapeutic targets to combat multidrug resistant A. baumannii infections.
Assuntos
Infecções por Acinetobacter/imunologia , Acinetobacter baumannii/imunologia , Complexo Antígeno L1 Leucocitário/imunologia , Pneumonia Bacteriana/imunologia , Zinco/imunologia , Infecções por Acinetobacter/tratamento farmacológico , Infecções por Acinetobacter/genética , Acinetobacter baumannii/genética , Acinetobacter baumannii/patogenicidade , Animais , Transporte Biológico Ativo , Carbapenêmicos/farmacologia , Modelos Animais de Doenças , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/genética , Farmacorresistência Bacteriana Múltipla/imunologia , Humanos , Pulmão/imunologia , Pulmão/patologia , Manganês/imunologia , Camundongos , Camundongos Knockout , Infiltração de Neutrófilos/genética , Infiltração de Neutrófilos/imunologia , Neutrófilos/imunologia , Neutrófilos/patologia , Pneumonia Bacteriana/genética , Pneumonia Bacteriana/patologiaRESUMO
Base propenals are products of the reaction of DNA with oxidants such as peroxynitrite and bleomycin. The most reactive base propenal, adenine propenal, is mutagenic in Escherichia coli and reacts with DNA to form covalent adducts; however, the reaction of adenine propenal with protein has not yet been investigated. A survey of the reaction of adenine propenal with amino acids revealed that lysine and cysteine form adducts, whereas histidine and arginine do not. N(ε)-Oxopropenyllysine, a lysine-lysine cross-link, and S-oxopropenyl cysteine are the major products. Comprehensive profiling of the reaction of adenine propenal with human serum albumin and the DNA repair protein, XPA, revealed that the only stable adduct is N(ε)-oxopropenyllysine. The most reactive sites for modification in human albumin are K190 and K351. Three sites of modification of XPA are in the DNA-binding domain, and two sites are subject to regulatory acetylation. Modification by adenine propenal dramatically reduces XPA's ability to bind to a DNA substrate.
Assuntos
Adenina/análogos & derivados , Albumina Sérica/química , Proteína de Xeroderma Pigmentoso Grupo A/química , Adenina/química , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Cisteína/química , Polarização de Fluorescência , Humanos , Lisina/química , Dados de Sequência Molecular , Peptídeos/análise , Peptídeos/química , Espectrometria de Massas em TandemRESUMO
Type 1 diabetes (T1D) affects three million Americans, with 80 new people diagnosed each day. T1D is currently uncurable and there is an urgent need to develop additional drug candidates to achieve the prevention of T1D. We propose AZD6738 (ATRi), an orally available drug currently in phases I and II of clinical trials for various cancers, as a novel candidate to prevent T1D. Based on previously reported findings of ATRi inducing cell death in rapidly proliferating T cells, we hypothesized that this drug would specifically affect self-antigen activated diabetogenic T cells. These cells, if left unchecked, could otherwise lead to the destruction of pancreatic ß cells, contributing to the development of T1D. This work demonstrates that increasing the duration of ATRi treatment provides extended protection against T1D onset. Remarkably, 5-week ATRi treatment prevented T1D in a robust adoptive transfer mouse model. Furthermore, the splenocytes of animals that received 5-week ATRi treatment did not transfer immune-mediated diabetes, while the splenocytes from control animal transferred the disease in 10 days. This work shows that ATRi prevents T1D by specifically inducing cell death in self-antigen activated, highly proliferative diabetogenic T cells through the induction of DNA damage, resulting in the inhibition of IFNγ production and proliferation. These findings support the consideration of repurposing ATRi for T1D prevention.
Assuntos
Antineoplásicos , Diabetes Mellitus Tipo 1 , Neoplasias , Animais , Camundongos , Humanos , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 1/prevenção & controle , Indóis , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , AutoantígenosRESUMO
ATR kinase is a central regulator of the DNA damage response (DDR) and cell cycle checkpoints. ATR kinase inhibitors (ATRi's) combine with radiation to generate CD8+ T cell-dependent responses in mouse models of cancer. We show that ATRi's induce cyclin-dependent kinase 1 (CDK1)-dependent origin firing across active replicons in CD8+ T cells activated ex vivo while simultaneously decreasing the activity of rate-limiting enzymes for nucleotide biosynthesis. These pleiotropic effects of ATRi induce deoxyuridine (dU) contamination in genomic DNA, R loops, RNA-DNA polymerase collisions, and interferon-α/ß (IFN-α/ß). Remarkably, thymidine rescues ATRi-induced dU contamination and partially rescues death and IFN-α/ß expression in proliferating CD8+ T cells. Thymidine also partially rescues ATRi-induced cancer cell death. We propose that ATRi-induced dU contamination contributes to dose-limiting leukocytopenia and inflammation in the clinic and CD8+ T cell-dependent anti-tumor responses in mouse models. We conclude that ATR is essential to limit dU contamination in genomic DNA and IFN-α/ß expression.
Assuntos
Linfócitos T CD8-Positivos , Proteína Quinase CDC2 , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Proteína Quinase CDC2/metabolismo , Morte Celular , Linhagem Celular Tumoral , DNA , Dano ao DNA , DNA Polimerase Dirigida por DNA/metabolismo , Desoxiuridina , Genômica , Interferon-alfa/metabolismo , Interferon beta , Camundongos , Nucleotídeos/metabolismo , Inibidores de Proteínas Quinases/farmacologia , RNA , Timidina/farmacologiaRESUMO
Nucleotide excision repair (NER) is essential for removing many types of DNA lesions from the genome, yet the mechanisms of NER in humans remain poorly understood. This review summarizes our current understanding of the structure, biochemistry, interaction partners, mechanisms, and disease-associated mutations of one of the critical NER proteins, XPA.
Assuntos
Reparo do DNA , DNA/metabolismo , Proteína de Xeroderma Pigmentoso Grupo A/metabolismo , Xeroderma Pigmentoso/genética , Sequência de Aminoácidos , Animais , DNA/química , Dano ao DNA , Humanos , Modelos Moleculares , Mutação , Ligação Proteica , Domínios Proteicos , Mapeamento de Interação de Proteínas , Estrutura Secundária de Proteína , Índice de Gravidade de Doença , Xeroderma Pigmentoso/metabolismo , Xeroderma Pigmentoso/patologia , Proteína de Xeroderma Pigmentoso Grupo A/química , Proteína de Xeroderma Pigmentoso Grupo A/genéticaRESUMO
DNA replication, damage response and repair require the coordinated action of multi-domain proteins operating within dynamic multi-protein machines that act upon the DNA substrate. These modular proteins contain flexible linkers of various lengths, which enable changes in the spatial distribution of the globular domains (architecture) that harbor their essential biochemical functions. This mobile architecture is uniquely suited to follow the evolving substrate landscape present over the course of the specific process performed by the multi-protein machinery. A fundamental advance in understanding of protein machinery is the realization of the pervasive role of dynamics. Not only is the machine undergoing dynamic transformations, but the proteins themselves are flexible and constantly adapting to the progression through the steps of the overall process. Within this dynamic context the activity of the constituent proteins must be coordinated, a role typically played by hub proteins. A number of important characteristics of modular proteins and concepts about the operation of dynamic machinery have been discerned. These provide the underlying basis for the action of the machinery that reads DNA, and responds to and repairs DNA damage. Here, we introduce a number of key characteristics and concepts, including the modularity of the proteins, linkage of weak binding sites, direct competition between sites, and allostery, using the well recognized hub protein replication protein A (RPA).