RESUMO
MTH1 (MutT homolog 1) or NUDT1 (Nudix Hydrolase 1), also known as oxidized purine nucleoside triphosphatase, has potential as a biomarker for monitoring cancer progression and quantifying target engagement for relevant therapies. In this study, we validate one MTH1 inhibitor TH287 as a PET MTH1 radiotracer. TH287 was radiolabeled with tritium and the binding of [3H]TH287 to MTH1 was evaluated in live glioblastoma cells (U251MG) through saturation and competitive binding assays, together with in vitro enzymatic assays. Furthermore, TH287 was radiolabeled with carbon-11 for in vivo microPET studies. Saturation binding assays show that [3H]TH287 has a dissociation constant (Kd) of 1.97 ± 0.18 nM, Bmax of 2676 ± 122 fmol/mg protein for U251MG cells, and nH of 0.98 ± 0.02. Competitive binding assays show that TH287 (Ki: 3.04 ± 0.14 nM) has a higher affinity for MTH1 in U251MG cells compared to another well studied MTH1 inhibitor: (S)-crizotinib (Ki: 153.90 ± 20.48 nM). In vitro enzymatic assays show that TH287 has an IC50 of 2.2 nM in inhibiting MTH1 hydrolase activity and a Ki of 1.3 nM from kinetics assays, these results are consistent with our radioligand binding assays. Furthermore, MicroPET imaging shows that [11C]TH287 gets into the brain with rapid clearance from the brain, kidney, and heart. The results presented here indicate that radiolabeled TH287 has favorable properties to be a useful tool for measuring MTH1 in vitro and for further evaluation for in vivo PET imaging MTH1 of brain tumors and other central nervous system disorders.
Assuntos
Biomarcadores Tumorais/isolamento & purificação , Enzimas Reparadoras do DNA/genética , Glioblastoma/diagnóstico por imagem , Monoéster Fosfórico Hidrolases/genética , Pirimidinas/farmacologia , Animais , Biomarcadores Tumorais/metabolismo , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Linhagem Celular Tumoral , Crizotinibe/farmacologia , Enzimas Reparadoras do DNA/antagonistas & inibidores , Enzimas Reparadoras do DNA/isolamento & purificação , Glioblastoma/genética , Glioblastoma/patologia , Coração/diagnóstico por imagem , Humanos , Rim/diagnóstico por imagem , Rim/metabolismo , Camundongos , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Monoéster Fosfórico Hidrolases/isolamento & purificação , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Pirimidinas/químicaRESUMO
Uracil-DNA glycosylases (UDGs) are conserved DNA-repair enzymes that can be found in many species, including herpesviruses. Since they play crucial roles for efficient viral DNA replication in herpesviruses, they have been considered as potential antiviral targets. In our previous work, Staphylococcus aureus SAUGI was identified as a DNA mimic protein that targets UDGs from S. aureus, human, Herpes simplex virus (HSV) and Epstein-Barr virus (EBV). Interestingly, SAUGI has the strongest inhibitory effects with EBVUDG. Here, we determined complex structures of SAUGI with EBVUDG and another γ-herpesvirus UDG from Kaposi's sarcoma-associated herpesvirus (KSHVUDG), which SAUGI fails to effectively inhibit. Structural analysis of the SAUGI/EBVUDG complex suggests that the additional interaction between SAUGI and the leucine loop may explain why SAUGI shows the highest binding capacity with EBVUDG. In contrast, SAUGI appears to make only partial contacts with the key components responsible for the compression and stabilization of the DNA backbone in the leucine loop extension of KSHVUDG. The findings in this study provide a molecular explanation for the differential inhibitory effects and binding strengths that SAUGI has on these two UDGs, and the structural basis of the differences should be helpful in developing inhibitors that would interfere with viral DNA replication.
Assuntos
Enzimas Reparadoras do DNA/química , Gammaherpesvirinae/enzimologia , Uracila-DNA Glicosidase/química , Substituição de Aminoácidos , Enzimas Reparadoras do DNA/isolamento & purificação , Enzimas Reparadoras do DNA/metabolismo , Replicação do DNA , Modelos Moleculares , Conformação Molecular , Ligação Proteica , Proteínas Recombinantes , Relação Estrutura-Atividade , Uracila-DNA Glicosidase/isolamento & purificação , Uracila-DNA Glicosidase/metabolismoRESUMO
We develop a low-background electrochemical biosensor for one-step detection of uracil DNA glycosylase (UDG) based on the host-guest interaction and iron-embedded nitrogen-rich carbon nanotube (Fe-N-C) that mimics enzyme-mediated electrocatalysis to achieve signal amplification. In this work, Fe-N-C is initially immobilized on a glassy carbon electrode, followed by the immobilization of ß-cyclodextrin (ß-CD). We construct the signal probes by assembling the methylene blue (MB)-labeled hairpin DNAs onto the surface of Au nanoparticles (AuNPs) to form the MB-hairpin/AuNP probes. Due to the steric effect of AuNPs and the stem-loop structure of hairpin DNA, MB is prevented from entering the cavity of ß-CD on the electrode. In contrast, UDG enables the removal of uracil from the Uâ¢A pairs in the stem of hairpin DNA probe to generate apurinic/apyrimidinic (AP) sites, leading to the assembly of MB-hairpin/AuNP probes on the electrode based on host-guest reaction between ß-CD and MB. Meanwhile, L-cysteine (RSH) is oxidized by O2 to disulfide L-cystine (RSSR) and H2O2. In the presence of H2O2, Fe-N-C catalyzes the oxidation of MB to generate an amplified electrochemical signal. Notably, the Fe-N-C-catalyzed oxidation of MB is mediated by the oxidation of RSH by O2 instead of external H2O2, greatly simplifying the experimental procedures and improving the electrochemical signal. Due to the introduction of host-guest recognition, this electrochemical biosensor displays a low-background signal and high signal-to-noise ratio, enabling the one-step sensitive measurement of UDG with a detection limit of 7.4â¯×â¯10-5 U mL-1. Moreover, this biosensor can measure UDG in crude cell extracts and screen the inhibitors, providing a new platform for biomedical research.
Assuntos
Técnicas Biossensoriais , Enzimas Reparadoras do DNA/isolamento & purificação , Reparo do DNA/genética , Técnicas Eletroquímicas , Enzimas Reparadoras do DNA/genética , Ouro/química , Peróxido de Hidrogênio/química , Ferro/química , Nanopartículas Metálicas/química , Azul de Metileno/química , Nanotubos de Carbono/químicaRESUMO
Human mutT homolog 1(MTH1), the oxidized dNTP pool sanitizer enzyme, has been reported to be highly expressed in various malignant tumors. However, the oncogenic role of MTH1 in gastric cancer remains to be determined. In the current study, we found that MTH1 was overexpressed in human gastric cancer tissues and cells. Using an in vitro MTH1 inhibitor screening system, the compounds available in our laboratory were screened and the small molecules containing 5-cyano-6-phenylpyrimidine structure were firstly found to show potently and specifically inhibitory effect on MTH1, especially compound MI-743 with IC50 = 91.44 ± 1.45 nM. Both molecular docking and target engagement experiments proved that MI-743 can directly bind to MTH1. Moreover, MI-743 could not only inhibit cell proliferation in up to 16 cancer cell lines, especially gastric cancer cells HGC-27 and MGC-803, but also significantly induce MTH1-related 8-oxo-dG accumulation and DNA damage. Furthermore, the growth of xenograft tumours derived by injection of MGC-803 cells in nude mice was also significantly inhibited by MI-743 treatment. Importantly, MTH1 knockdown by siRNA in those two gastric cancer cells exhibited the similar findings. Our findings indicate that MTH1 is highly expressed in human gastric cancer tissues and cell lines. Small molecule MI-743 with 5-cyano-6-phenylpyrimidine structure may serve as a novel lead compound targeting the overexpressed MTH1 for gastric cancer treatment.
Assuntos
8-Hidroxi-2'-Desoxiguanosina/metabolismo , Antineoplásicos/farmacologia , Enzimas Reparadoras do DNA/antagonistas & inibidores , Enzimas Reparadoras do DNA/metabolismo , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Monoéster Fosfórico Hidrolases/metabolismo , Neoplasias Gástricas/metabolismo , Animais , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Dano ao DNA/efeitos dos fármacos , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/isolamento & purificação , Feminino , Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Simulação de Acoplamento Molecular , Mutação , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/isolamento & purificação , Neoplasias Gástricas/genética , Transplante HeterólogoRESUMO
Human MutT homolog 1 (MTH1) hydrolyses oxidised nucleotide triphosphates, thereby preventing them from being incorporated into DNA; MTH1 has been found to be elevated in many types of cancers, including lung, stomach cancer, melanoma and breast cancer. Thus, tumourtargeted hMTH1 may be valuable for developing novel anticancer therapies. In the present study, we prepared human MTH1 protein and its monoclonal antibody (mAb). The hMTH1 gene was cloned into the prokaryotic expression vector pET28a and optimally expressed in the E. coli Transetta (DE3) strain. Using an NiNTA column and a G50 gel filtration column, 20.1 mg of active hMTH1 was obtained from 1,000 ml of bacterial culture, and the purity was over 98%, as detected by highperformance liquid chromatography (HPLC). The half maximal inhibitory concentration (IC50) of TH287 (hMTH1 inhibitor) was determined to be 3.53±0.47 nM using the recombinant hMTH1 protein (rhMTH1). The enzyme activity assay showed the Michaelis constant (Km) and the catalytic constant (kcat) of the protein were 106.13±48.83 µM and 3.64±0.58 sec1, respectively. The antihMTH1 mAb was obtained via the hybridoma technique and validated by western blot analysis. In addition, an immunofluorescence assay (IFA) and ELISA determined that the mAb could efficiently bind to natural hMTH1 expressed on the human breast cancer cell line MCF7. Taken together, the results showed the rhMTH1 is an active protein and has practical applications for inhibitor selection, and our prepared hMTH1 mAb will provide a valuable tool for the further characterisation of hMTH1 and antitumour medicinal development in future.
Assuntos
Anticorpos Monoclonais/imunologia , Enzimas Reparadoras do DNA/imunologia , Enzimas Reparadoras do DNA/metabolismo , Desenvolvimento de Medicamentos/métodos , Monoéster Fosfórico Hidrolases/imunologia , Monoéster Fosfórico Hidrolases/metabolismo , Anticorpos Monoclonais/isolamento & purificação , Linhagem Celular Tumoral , Clonagem Molecular , Enzimas Reparadoras do DNA/antagonistas & inibidores , Enzimas Reparadoras do DNA/isolamento & purificação , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Ensaios Enzimáticos/métodos , Humanos , Oxirredução , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Monoéster Fosfórico Hidrolases/isolamento & purificação , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
PAXX is a recently identified component of the nonhomologous end joining (NHEJ) DNA repair pathway. The molecular mechanisms of PAXX action remain largely unclear. Here we characterise the interactomes of PAXX and its paralogs, XLF and XRCC4, to show that these factors share the ability to interact with DNA polymerase λ (Pol λ), stimulate its activity and are required for recruitment of Pol λ to laser-induced DNA damage sites. Stimulation of Pol λ activity by XRCC4 paralogs requires a direct interaction between the SP/8 kDa domain of Pol λ and their N-terminal head domains to facilitate recognition of the 5' end of substrate gaps. Furthermore, PAXX and XLF collaborate with Pol λ to promote joining of incompatible DNA ends and are redundant in supporting Pol λ function in vivo. Our findings identify Pol λ as a novel downstream effector of PAXX function and show XRCC4 paralogs act in synergy to regulate polymerase activity in NHEJ.
Assuntos
Reparo do DNA por Junção de Extremidades/fisiologia , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/isolamento & purificação , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/isolamento & purificação , Células HEK293 , Humanos , Lasers/efeitos adversos , Mutagênese Sítio-Dirigida , Ligação Proteica/fisiologia , Domínios Proteicos/fisiologia , Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas/fisiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Espectrometria de Massas em Tandem/métodosRESUMO
DNA end resection initiates the largely accurate repair of DNA double-strand breaks (DSBs) by homologous recombination. Specifically, recombination requires the formation of 3' overhangs at DSB sites, which is carried out by nucleases that specifically degrade 5'-terminated DNA. In most cases, DNA end resection is a two-step process, comprising of initial short-range followed by more processive long-range resection. In this chapter, we describe selected assays that reconstitute both the short- and long-range pathways. First, we define methods to study the exonuclease and endonuclease activities of the MRE11-RAD50-NBS1 (MRN) complex in conjunction with phosphorylated cofactor CtIP. This reaction is particularly important to initiate processing of DNA breaks and to recruit components belonging to the subsequent long-range pathway. Next, we describe assays that reconstitute the concerted reactions of Bloom (BLM) or Werner (WRN) helicases that function together with the DNA2 nuclease-helicase, and which are as a complex capable to resect DNA of kilobases in length. The reconstituted reactions allow us to understand how the resection pathways function at the molecular level. The assays will be invaluable to define regulatory mechanisms and to identify inhibitory compounds, which may be valuable in cancer therapy.
Assuntos
Técnicas de Cultura de Células/métodos , Quebras de DNA de Cadeia Dupla , Ensaios Enzimáticos/métodos , Proteínas Recombinantes/isolamento & purificação , Reparo de DNA por Recombinação , Hidrolases Anidrido Ácido , Animais , Baculoviridae/genética , Soluções Tampão , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/metabolismo , Técnicas de Cultura de Células/instrumentação , Proteínas de Ciclo Celular/isolamento & purificação , Proteínas de Ciclo Celular/metabolismo , DNA Helicases/isolamento & purificação , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/isolamento & purificação , Enzimas Reparadoras do DNA/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Eletroforese em Gel de Poliacrilamida/instrumentação , Eletroforese em Gel de Poliacrilamida/métodos , Endodesoxirribonucleases , Ensaios Enzimáticos/instrumentação , Humanos , Proteína Homóloga a MRE11/isolamento & purificação , Proteína Homóloga a MRE11/metabolismo , Proteínas Nucleares/isolamento & purificação , Proteínas Nucleares/metabolismo , Oligonucleotídeos/metabolismo , RecQ Helicases/isolamento & purificação , RecQ Helicases/metabolismo , Proteínas Recombinantes/metabolismo , Proteína de Replicação A/isolamento & purificação , Proteína de Replicação A/metabolismo , Células Sf9 , Spodoptera , Transfecção/métodos , Helicase da Síndrome de Werner/isolamento & purificação , Helicase da Síndrome de Werner/metabolismoRESUMO
Exocyclic ethenobases are highly mutagenic DNA lesions strongly implicated in inflammation and vinyl chloride-induced carcinogenesis. While the alkyladenine DNA glycosylase, AAG (or MPG), binds the etheno lesions 1,N(6)-ethenoadenine (ÉA) and 3,N(4)-ethenocytosine (ÉC) with high affinity, only ÉA can be excised to initiate base excision repair. Here, we discover that the human AlkB homolog 2 (ALKBH2) dioxygenase enzyme catalyzes direct reversal of ÉC lesions in both double- and single-stranded DNA with comparable efficiency to canonical ALKBH2 substrates. Notably, we find that in vitro, the non-enzymatic binding of AAG to ÉC specifically blocks ALKBH2-catalyzed repair of ÉC but not that of methylated ALKBH2 substrates. These results identify human ALKBH2 as a repair enzyme for mutagenic ÉC lesions and highlight potential consequences for substrate-binding overlap between the base excision and direct reversal DNA repair pathways.
Assuntos
Citosina/análogos & derivados , DNA Glicosilases/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Reparo do DNA , Dioxigenases/metabolismo , Homólogo AlkB 2 da Dioxigenase Dependente de alfa-Cetoglutarato , Citosina/metabolismo , Dano ao DNA , Enzimas Reparadoras do DNA/isolamento & purificação , Enzimas de Restrição do DNA/metabolismo , Dioxigenases/isolamento & purificação , Humanos , Especificidade por SubstratoRESUMO
UNLABELLED: Proteins tightly bound to DNA (TBP) comprise a group of proteins that remain bound to DNA even after harsh deproteinization procedures. The amount of these proteins is 20-100 µg for mg of DNA depending on eukaryotic source. This experimental paper examines the possibility to use some TBP for clinical biomarker discovery, e.g. for identification of prognostic and diagnostic cancer markers. The main aim of this study was to designate differences between tightly DNA binding protein patterns extracted from rat liver and rat experimental hepatomas (Zajdela ascites hepatoma and hepatoma G-27) and to evaluate possibility that some of these proteins may be used as biomarkers for cell cancer transformation. METHODS: We used proteomics aproach as a tool for comparison of pattern of TBP from rat experimental hepatomas and normal liver cells. Combination of 2DE fractionation with mass spectrometry (MALDI TOF-MS) suitable for parallel profiling of complex TBP mixtures. RESULTS: Intriguingly 2DE protein maps of TBP from rat liver and rat experimental hepatomas (Zajdela acites hepatoma and hepatoma G-27) were quite different. We identified 9 proteins, some of them shared in all TBP patterns. Among identified tightly bound to DNA proteins there were three proteins considered as nuclear matrix proteins (lamin B1, scaffold attachment factor B1, heterogeneous nuclear ribonucleoprotein). Also we identified DNA repair protein RAD50, coiled-coil domain-containing protein 41, structural maintenance of chromosomes protein1A and some ATP -dependent RNA helicases indicating that TBP are of interest with respect to their potential involvement in the topological organization and/ or function of genomic DNA. CONCLUSIONS: We suppose that proteomic approach for TBP identification may be promising in development of biomarkers, also obtained results may be valuable for further understanding TBP functions in genome.
Assuntos
Biomarcadores Tumorais/isolamento & purificação , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Neoplasias Hepáticas Experimentais/metabolismo , Animais , Biomarcadores Tumorais/metabolismo , Enzimas Reparadoras do DNA/isolamento & purificação , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/genética , Fígado/metabolismo , Neoplasias Hepáticas Experimentais/genética , Matriz Nuclear , Proteínas Associadas à Matriz Nuclear/isolamento & purificação , Proteínas Associadas à Matriz Nuclear/metabolismo , Prognóstico , Ligação Proteica , Proteômica , RNA Helicases/isolamento & purificação , RNA Helicases/metabolismo , Ratos , Ratos Wistar , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The ability to monitor and characterize DNA mismatch repair activity in various mammalian cells is important for understanding mechanisms involved in mutagenesis and tumorigenesis. Since mismatch repair proteins recognize mismatches containing both normal and chemically altered or damaged bases, in vitro assays must accommodate a variety of mismatches in different sequence contexts. Here we describe the construction of DNA mismatch substrates containing G:T or O(6)meG:T mismatches, the purification of recombinant native human MutSα (MSH2-MSH6) and MutLα (MLH1-PMS2) proteins, and in vitro mismatch repair and excision assays that can be adapted to study mismatch repair in nuclear extracts from mismatch repair proficient and deficient cells.
Assuntos
Reparo de Erro de Pareamento de DNA , Enzimas Reparadoras do DNA/metabolismo , Proteínas Recombinantes/metabolismo , Sequência de Bases , Linhagem Celular , Enzimas Reparadoras do DNA/química , Enzimas Reparadoras do DNA/isolamento & purificação , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Humanos , Proteínas MutL , Polinucleotídeos/química , Polinucleotídeos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
The aim of this study was to characterize rhizobial isolates from Cratylia mollis Mart. ex Benth, Calliandra depauperata Benth. and Mimosa tenuiflora (Willd.) Poir. by means of rhizobial colonies morphology and restriction analysis of the 16S ribosomal gene (16S rDNA-ARDRA). Nodules were collected in the field and from plants cultivated in a greenhouse experiment using Caatinga soil samples. Sixty seven isolates were described by morphological analysis. Forty seven representative isolates were used for ARDRA analysis using seven restriction enzymes. We observed high diversity of both slow and fast-growing rhizobia that formed three morpho-physiological clusters. A few fast-growing isolates formed a group of strains of the Bradyrhizobium type; however, most of them diverged from the B. japonicum and B. elkanii species. Cratylia mollis nodule isolates were the most diverse, while all Mimosa tenuiflora isolates displayed fast growth with no pH change and were clustered into groups bearing 100 percent similarity, according to ARDRA results.
Assuntos
Ativação Enzimática/genética , Técnicas In Vitro , Rhizobiaceae/citologia , Rhizobiaceae/genética , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/isolamento & purificação , Genes de Plantas , Variação Genética , Reação em Cadeia da Polimerase , Rhizobiaceae/crescimento & desenvolvimentoRESUMO
Nonhomologous end joining (NHEJ) is the major pathway for the repair of DNA double strand breaks (DSBs) in human cells. NHEJ requires the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), Ku70, Ku80, XRCC4, DNA ligase IV and Artemis, as well as DNA polymerases mu and lambda and polynucleotide kinase. Recent studies have identified an additional participant, XLF, for XRCC4-like factor (also called Cernunnos), which interacts with the XRCC4-DNA ligase IV complex and stimulates its activity in vitro, however, its precise role in the DNA damage response is not fully understood. Since the protein kinase activity of DNA-PKcs is required for NHEJ, we asked whether XLF might be a physiological target of DNA-PK. Here, we have identified two major in vitro DNA-PK phosphorylation sites in the C-terminal region of XLF, serines 245 and 251. We show that these represent the major phosphorylation sites in XLF in vivo and that serine 245 is phosphorylated in vivo by DNA-PK, while serine 251 is phosphorylated by Ataxia-Telangiectasia Mutated (ATM). However, phosphorylation of XLF did not have a significant effect on the ability of XLF to interact with DNA in vitro or its recruitment to laser-induced DSBs in vivo. Similarly, XLF in which the identified in vivo phosphorylation sites were mutated to alanine was able to complement the DSB repair defect as well as radiation sensitivity in XLF-deficient 2BN cells. We conclude that phosphorylation of XLF at these sites does not play a major role in the repair of IR-induced DSBs in vivo.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Enzimas Reparadoras do DNA/metabolismo , Reparo do DNA , Proteína Quinase Ativada por DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Sequência de Aminoácidos , Aminoácidos/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Sobrevivência Celular/efeitos da radiação , DNA/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Reparo do DNA/efeitos da radiação , Enzimas Reparadoras do DNA/química , Enzimas Reparadoras do DNA/isolamento & purificação , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/isolamento & purificação , Células HeLa , Humanos , Dados de Sequência Molecular , Proteínas Mutantes/metabolismo , Fosforilação/efeitos da radiação , Fosfosserina/metabolismo , Ligação Proteica/efeitos da radiação , Estrutura Terciária de Proteína , Transporte Proteico/efeitos da radiação , Radiação Ionizante , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
It has been hypothesized that a replication associated repair pathway operates on base damage and single strand breaks (SSB) at replication forks. In this study, we present the isolation from the nuclei of human cycling cells of a multiprotein complex containing most of the essential components of base excision repair (BER)/SSBR, including APE1, UNG2, XRCC1 and POLbeta, DNA PK, replicative POLalpha, delta and epsilon, DNA ligase 1 and cell cycle regulatory protein cyclin A. Co-immunoprecipitation revealed that in this complex DNA repair proteins are physically associated to cyclin A and to DNA replication proteins including MCM7. This complex is endowed with DNA polymerase and protein kinase activity and is able to perform BER of uracil and AP sites. This finding suggests that a preassembled DNA repair machinery is constitutively active in cycling cells and is ready to be recruited at base damage and breaks occurring at replication forks.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Reparo do DNA , Replicação do DNA , Animais , Proteínas de Ciclo Celular/isolamento & purificação , Ciclina A/isolamento & purificação , Ciclina A/metabolismo , Enzimas Reparadoras do DNA/isolamento & purificação , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/isolamento & purificação , DNA Polimerase Dirigida por DNA/metabolismo , Células HeLa , Humanos , Imunoprecipitação , Camundongos , Componente 7 do Complexo de Manutenção de Minicromossomo , Proteínas Nucleares/isolamento & purificação , Proteínas Nucleares/metabolismoRESUMO
Ataxia-telangiectasia mutated (ATM) is a serine-threonine kinase that is activated by DNA double strand breaks to phosphorylate many cellular proteins involved in cell cycle regulation and DNA repair. We have shown previously that the activation of ATM can be reconstituted in an in vitro system using recombinant human ATM. In this system, ATM activity is dependent on the Mre11/Rad50/Nbs1 (MRN) complex and linear DNA, similar to requirements observed in human cells. This chapter describes methods used for the overexpression and purification of human ATM and MRN, as well as a protocol for in vitro kinase assays.
Assuntos
Proteínas de Ciclo Celular , Enzimas Reparadoras do DNA , Proteínas de Ligação a DNA , Proteínas Nucleares , Proteínas Serina-Treonina Quinases , Proteínas Supressoras de Tumor , Hidrolases Anidrido Ácido , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/isolamento & purificação , Proteínas de Ciclo Celular/metabolismo , Dano ao DNA , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/isolamento & purificação , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Humanos , Proteína Homóloga a MRE11 , Complexos Multiproteicos , Proteínas Nucleares/genética , Proteínas Nucleares/isolamento & purificação , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/isolamento & purificação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/isolamento & purificação , Proteínas Supressoras de Tumor/metabolismoRESUMO
The mismatch repair system (MMR) recognizes and corrects mismatched or unpaired bases caused mainly by DNA polymerase, and contributes to the fidelity of DNA replication in living cells. In Escherichia coli, the MutHLS system is known to function in MMR, and homologues of MutS and MutL are widely conserved in almost all organisms. However, the MutH endonuclease has not been found in the majority of organisms. Such organisms, including Thermus thermophilus HB8, often possess the so-called MutS2 protein, which is highly homologous to MutS but contains an extra C-terminal stretch. To elucidate the function of MutS2, we overexpressed and purified T. thermophilus MutS2 (ttMutS2). ttMutS2 demonstrated the ability to bind double-stranded (ds) DNA, but, unlike ttMutS, ttMutS2 showed no specificity for mismatched duplexes. ttMutS2 ATPase activity was also detected and was stimulated by dsDNA. Our results also showed that ttMutS2 incises dsDNA. ttMutS2 incises not only oligo dsDNA but also plasmid DNA, suggesting that ttMutS2 possesses an endonuclease activity. At low concentrations, the incision activity was not retained, but was promoted by T. thermophilus MutL.