RESUMO
Polynucleotide kinase/phosphatase (PNKP) and X-ray repair cross-complementing 1 (XRCC1) are key proteins in the single-strand DNA break repair pathway. Phosphorylated XRCC1 stimulates PNKP by binding to its forkhead-associated (FHA) domain, whereas nonphosphorylated XRCC1 stimulates PNKP by interacting with the PNKP catalytic domain. Here, we have further studied the interactions between these two proteins, including two variants of XRCC1 (R194W and R280H) arising from single-nucleotide polymorphisms (SNPs) that have been associated with elevated cancer risk in some reports. We observed that interaction of the PNKP FHA domain with phosphorylated XRCC1 extends beyond the immediate, well-characterized phosphorylated region of XRCC1 (residues 515-526). We also found that an XRCC1 fragment, comprising residues 166-436, binds tightly to PNKP and DNA and efficiently activates PNKP's kinase activity. However, interaction of either of the SNP-derived variants of this fragment with PNKP was considerably weaker, and their stimulation of PNKP was severely reduced, although they still could bind DNA effectively. Laser microirradiation revealed reduced recruitment of PNKP to damaged DNA in cells expressing either XRCC1 variant compared with PNKP recruitment in cells expressing WT XRCC1 even though WT and variant XRCC1s were equally efficient at localizing to the damaged DNA. These findings suggest that the elevated risk of cancer associated with these XRCC1 SNPs reported in some studies may be due in part to the reduced ability of these XRCC1 variants to recruit PNKP to damaged DNA.
Assuntos
Enzimas Reparadoras do DNA/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Polimorfismo de Nucleotídeo Único , Domínios e Motivos de Interação entre Proteínas , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/genética , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/metabolismo , Animais , Células CHO , Cricetulus , Dano ao DNA , Enzimas Reparadoras do DNA/química , Fosfotransferases (Aceptor do Grupo Álcool)/química , Mapas de Interação de Proteínas , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/químicaRESUMO
The scaffold protein X-ray repair cross-complementing 1 (XRCC1) interacts with multiple enzymes involved in DNA base excision repair and single-strand break repair (SSBR) and is important for genetic integrity and normal neurological function. One of the most important interactions of XRCC1 is that with polynucleotide kinase/phosphatase (PNKP), a dual-function DNA kinase/phosphatase that processes damaged DNA termini and that, if mutated, results in ataxia with oculomotor apraxia 4 (AOA4) and microcephaly with early-onset seizures and developmental delay (MCSZ). XRCC1 and PNKP interact via a high-affinity phosphorylation-dependent interaction site in XRCC1 and a forkhead-associated domain in PNKP. Here, we identified using biochemical and biophysical approaches a second PNKP interaction site in XRCC1 that binds PNKP with lower affinity and independently of XRCC1 phosphorylation. However, this interaction nevertheless stimulated PNKP activity and promoted SSBR and cell survival. The low-affinity interaction site required the highly conserved Rev1-interacting region (RIR) motif in XRCC1 and included three critical and evolutionarily invariant phenylalanine residues. We propose a bipartite interaction model in which the previously identified high-affinity interaction acts as a molecular tether, holding XRCC1 and PNKP together and thereby promoting the low-affinity interaction identified here, which then stimulates PNKP directly.
Assuntos
Quebras de DNA de Cadeia Simples , Enzimas Reparadoras do DNA/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Modelos Moleculares , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação , Ensaio Cometa , Sequência Conservada , Enzimas Reparadoras do DNA/química , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Humanos , Cinética , Mutação , Estresse Oxidativo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Proteína 1 Complementadora Cruzada de Reparo de Raio-XRESUMO
The termini of DNA strand breaks induced by internal and external factors often require processing before missing nucleotides can be replaced by DNA polymerases and the strands rejoined by DNA ligases. Polynucleotide kinase/phosphatase (PNKP) serves a crucial role in the repair of DNA strand breaks by catalyzing the restoration of 5'-phosphate and 3'-hydroxyl termini. It participates in several DNA repair pathways through interactions with other DNA repair proteins, notably XRCC1 and XRCC4. Recent studies have highlighted the physiological importance of PNKP in maintaining the genomic stability of normal tissues, particularly developing neural cells, as well as enhancing the resistance of cancer cells to genotoxic therapeutic agents.
Assuntos
Quebras de DNA , Enzimas Reparadoras do DNA/metabolismo , Reparo do DNA , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Sequência de Aminoácidos , Animais , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Ligação Proteica , Homologia de Sequência de Aminoácidos , Proteína 1 Complementadora Cruzada de Reparo de Raio-XRESUMO
Human polynucleotide kinase/phosphatase (PNKP) is a dual specificity 5'-DNA kinase/3'-DNA phosphatase, with roles in base excision repair, DNA single-strand break repair and non-homologous end joining (NHEJ); yet precisely how PNKP functions in the repair of DNA double strand breaks (DSBs) remains unclear. We demonstrate that PNKP is phosphorylated by the DNA-dependent protein kinase (DNA-PK) and ataxia-telangiectasia mutated (ATM) in vitro. The major phosphorylation site for both kinases was serine 114, with serine 126 being a minor site. Ionizing radiation (IR)-induced phosphorylation of cellular PNKP on S114 was ATM dependent, whereas phosphorylation of PNKP on S126 required both ATM and DNA-PK. Inactivation of DNA-PK and/or ATM led to reduced PNKP at DNA damage sites in vivo. Cells expressing PNKP with alanine or aspartic acid at serines 114 and 126 were modestly radiosensitive and IR enhanced the association of PNKP with XRCC4 and DNA ligase IV; however, this interaction was not affected by mutation of PNKP phosphorylation sites. Purified PNKP protein with mutation of serines 114 and 126 had decreased DNA kinase and DNA phosphatase activities and reduced affinity for DNA in vitro. Together, our results reveal that IR-induced phosphorylation of PNKP by ATM and DNA-PK regulates PNKP function at DSBs.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Enzimas Reparadoras do DNA/metabolismo , Proteína Quinase Ativada por DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Enzimas Reparadoras do DNA/química , Enzimas Reparadoras do DNA/genética , Células HeLa , Humanos , Mutação , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Tolerância a Radiação , Radiação Ionizante , Serina/metabolismoRESUMO
The XRCC4-like factor (XLF)-XRCC4 complex is essential for nonhomologous end joining, the major repair pathway for DNA double strand breaks in human cells. Yet, how XLF binds XRCC4 and impacts nonhomologous end joining functions has been enigmatic. Here, we report the XLF-XRCC4 complex crystal structure in combination with biophysical and mutational analyses to define the XLF-XRCC4 interactions. Crystal and solution structures plus mutations characterize alternating XRCC4 and XLF head domain interfaces forming parallel super-helical filaments. XLF Leu-115 ("Leu-lock") inserts into a hydrophobic pocket formed by XRCC4 Met-59, Met-61, Lys-65, Lys-99, Phe-106, and Leu-108 in synergy with pseudo-symmetric ß-zipper hydrogen bonds to drive specificity. XLF C terminus and DNA enhance parallel filament formation. Super-helical XLF-XRCC4 filaments form a positively charged channel to bind DNA and align ends for efficient ligation. Collective results reveal how human XLF and XRCC4 interact to bind DNA, suggest consequences of patient mutations, and support a unified molecular mechanism for XLF-XRCC4 stimulation of DNA ligation.
Assuntos
Quebras de DNA de Cadeia Dupla , Enzimas Reparadoras do DNA/química , Enzimas Reparadoras do DNA/metabolismo , Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Linhagem Celular , Cristalografia por Raios X , DNA/química , DNA/genética , DNA/metabolismo , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Humanos , Ligação Proteica/fisiologia , Estrutura Quaternária de Proteína , Estrutura Secundária de ProteínaRESUMO
XRCC1 plays a central role in mammalian single-strand break repair. Although it has no enzymatic activity of its own, it stimulates the activities of polynucleotide kinase/phosphatase (PNKP), and this function is enhanced by protein kinase CK2 mediated phosphorylation of XRCC1. We have previously shown that non-phosphorylated XRCC1 stimulates the kinase activity of PNKP by increasing the turnover of PNKP. Here we extend our analysis of the XRCC1-PNKP interaction taking into account the phosphorylation of XRCC1. We demonstrate that phosphorylated and non-phosphorylated XRCC1 interact with different regions of PNKP. Phosphorylated XRCC1 binds with high affinity (K(d) = 3.5 nM and 1 : 1 stoichiometry) to the forkhead associated (FHA) domain, while non-phosphorylated XRCC1 binds to the catalytic domain of PNKP with lower affinity (K(d) = 43.0 nM and 1 : 1 stoichiometry). Under conditions of limited enzyme concentration both forms of XRCC1 enhance the activities of PNKP, but the effect is more pronounced with phosphorylated XRCC1, particularly for the kinase activity of PNKP. The stimulatory effect of phosphorylated XRCC1 on PNKP can be totally inhibited by the presence of excess FHA domain polypeptide, but non-phosphorylated XRCC1 is not susceptible to competition by the FHA domain. Thus, XRCC1 can stimulate PNKP by two independent mechanisms.
Assuntos
Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Sítios de Ligação , Ligação Competitiva , DNA/metabolismo , Enzimas Reparadoras do DNA/química , Proteínas de Ligação a DNA/química , Humanos , Espectrometria de Massas , Peptídeos/metabolismo , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/química , Domínios e Motivos de Interação entre Proteínas , Proteína 1 Complementadora Cruzada de Reparo de Raio-XRESUMO
Inhibition of DNA repair enzymes is an attractive target for increasing the efficacy of DNA damaging chemotherapies. The ERCC1-XPF heterodimer is a key endonuclease in numerous single and double strand break repair processes, and inhibition of the heterodimerization has previously been shown to sensitize cancer cells to DNA damage. In this work, the previously reported ERCC1-XPF inhibitor 4 was used as the starting point for an in silico study of further modifications of the piperazine side-chain. A selection of the best scoring hits from the in silico screen were synthesized using a late stage functionalization strategy which should allow for further iterations of this class of inhibitors to be readily synthesized. Of the synthesized compounds, compound 6 performed the best in the in vitro fluorescence based endonuclease assay. The success of compound 6 in inhibiting ERCC1-XPF endonuclease activity in vitro translated well to cell-based assays investigating the inhibition of nucleotide excision repair and disruption of heterodimerization. Subsequently compound 6 was shown to sensitize HCT-116 cancer cells to treatment with UVC, cyclophosphamide, and ionizing radiation. This work serves as an important step towards the synergistic use of DNA repair inhibitors with chemotherapeutic drugs.
RESUMO
Polynucleotide Kinase-Phosphatase (PNKP) is a bifunctional enzyme that possesses both DNA 3'-phosphatase and DNA 5'-kinase activities, which are required for processing termini of single- and double-strand breaks generated by reactive oxygen species (ROS), ionizing radiation and topoisomerase I poisons. Even though PNKP is central to DNA repair, there have been no reports linking PNKP mutations in a Microcephaly, Seizures, and Developmental Delay (MSCZ) patient to cancer. Here, we characterized the biochemical significance of 2 germ-line point mutations in the PNKP gene of a 3-year old male with MSCZ who presented with a high-grade brain tumor (glioblastoma multiforme) within the cerebellum. Functional and biochemical studies demonstrated these PNKP mutations significantly diminished DNA kinase/phosphatase activities, altered its cellular distribution, caused defective repair of DNA single/double stranded breaks, and were associated with a higher propensity for oncogenic transformation. Our findings indicate that specific PNKP mutations may contribute to tumor initiation within susceptible cells in the CNS by limiting DNA damage repair and increasing rates of spontaneous mutations resulting in pediatric glioma associated driver mutations such as ATRX and TP53.
Assuntos
Neoplasias Encefálicas , Microcefalia , Neoplasias Encefálicas/genética , Criança , Pré-Escolar , Reparo do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Humanos , Masculino , Microcefalia/genética , Mutação , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Convulsões/genéticaRESUMO
The small molecule, 2-(1-hydroxyundecyl)-1-(4-nitrophenylamino)-6-phenyl-6,7a-dihydro-1H-pyrrolo[3,4-b]pyridine-5,7(2H,4aH)-dione (A12B4C3), is a potent inhibitor of the phosphatase activity of human polynucleotide kinase/phosphatase (PNKP) in vitro. Kinetic analysis revealed that A12B4C3 acts as a noncompetitive inhibitor, and this was confirmed by fluorescence quenching, which showed that the inhibitor can form a ternary complex with PNKP and a DNA substrate, i.e. A12B4C3 does not prevent DNA from binding to the phosphatase DNA binding site. Conformational analysis using circular dichroism, UV difference spectroscopy, and fluorescence resonance energy transfer all indicate that A12B4C3 disrupts the secondary structure of PNKP. Investigation of the potential site of binding of A12B4C3 to PNKP using site-directed mutagenesis pointed to interaction between Trp(402) of PNKP and the inhibitor. Cellular studies revealed that A12B4C3 sensitizes A549 human lung cancer cells to the topoisomerase I poison, camptothecin, but not the topoisomerase II poison, etoposide, in a manner similar to small interfering RNA against PNKP. A12B4C3 also inhibits the repair of DNA single and double strand breaks following exposure of cells to ionizing radiation, but does not inhibit two other key strand-break repair enzymes, DNA polymerase beta or DNA ligase III, providing additional evidence that PNKP is the cellular target of the inhibitor.
Assuntos
Enzimas Reparadoras do DNA/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Piperidinas/farmacologia , Pirróis/farmacologia , Trifosfato de Adenosina/metabolismo , Antineoplásicos Fitogênicos/farmacologia , Sítios de Ligação , Camptotecina/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Dicroísmo Circular , DNA Ligase Dependente de ATP , DNA Ligases/antagonistas & inibidores , DNA Ligases/metabolismo , DNA Polimerase beta/antagonistas & inibidores , DNA Polimerase beta/metabolismo , Reparo do DNA/efeitos dos fármacos , Enzimas Reparadoras do DNA/química , Enzimas Reparadoras do DNA/genética , Etoposídeo/farmacologia , Transferência Ressonante de Energia de Fluorescência , Humanos , Neoplasias Pulmonares/metabolismo , Mutagênese Sítio-Dirigida , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Espectroscopia Fotoeletrônica , Proteínas de Ligação a Poli-ADP-Ribose , Conformação Proteica , Pirróis/química , Radiossensibilizantes/farmacologia , Células Tumorais Cultivadas , Proteínas de XenopusRESUMO
XRCC4 plays a crucial role in the nonhomologous end joining (NHEJ) pathway of DNA double-strand break repair acting as a scaffold protein that recruits other NHEJ proteins to double-strand breaks. Phosphorylation of XRCC4 by protein kinase CK2 promotes a high affinity interaction with the forkhead-associated domain of the end-processing enzyme polynucleotide kinase/phosphatase (PNKP). Here we reveal that unphosphorylated XRCC4 also interacts with PNKP through a lower affinity interaction site within the catalytic domain and that this interaction stimulates the turnover of PNKP. Unexpectedly, CK2-phosphorylated XRCC4 inhibited PNKP activity. Moreover, the XRCC4·DNA ligase IV complex also stimulated PNKP enzyme turnover, and this effect was independent of the phosphorylation of XRCC4 at threonine 233. Our results reveal that CK2-mediated phosphorylation of XRCC4 can have different effects on PNKP activity, with implications for the roles of XRCC4 and PNKP in NHEJ.
Assuntos
Enzimas Reparadoras do DNA/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Caseína Quinase II/genética , Caseína Quinase II/metabolismo , DNA/genética , DNA/metabolismo , DNA Ligases/genética , DNA Ligases/metabolismo , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Células HeLa , Humanos , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
Phosphatase and TENsin homolog deleted on chromosome 10 (PTEN) is a major tumor-suppressor protein that is lost in up to 75% of aggressive colorectal cancers (CRC). The co-depletion of PTEN and a DNA repair protein, polynucleotide kinase 3'-phosphatase (PNKP), has been shown to lead to synthetic lethality in several cancer types including CRC. This finding inspired the development of novel PNKP inhibitors as potential new drugs against PTEN-deficient CRC. Here, we report on the in vitro and in vivo evaluation of a nano-encapsulated potent, but poorly water-soluble lead PNKP inhibitor, A83B4C63, as a new targeted therapeutic for PTEN-deficient CRC. Our data confirmed the binding of A83B4C63, as free or nanoparticle (NP) formulation, to intracellular PNKP using the cellular thermal shift assay (CETSA), in vitro and in vivo. Dose escalating toxicity studies in healthy CD-1 mice, based on measurement of animal weight changes and biochemical blood analysis, revealed the safety of both free and nano-encapsulated A83B4C63, at assessed doses of ≤50 mg/kg. Nano-carriers of A83B4C63 effectively inhibited the growth of HCT116/PTEN-/- xenografts in NIH-III nude mice following intravenous (IV) administration, but not that of wild-type HCT116/PTEN+/+ xenografts. This was in contrast to IV administration of A83B4C63 solubilized with the aid of Cremophor EL: Ethanol (CE), which led to similar tumor growth to that of formulation excipients (NP or CE without drug) or 5% dextrose. This observation was attributed to the higher levels of A83B4C63 delivered to tumor tissue by its NP formulation. Our data provide evidence for the success of NPs of A83B4C63, as novel synthetically lethal nano-therapeutics in the treatment of PTEN-deficient CRC. This research also highlights the potential of successful application of nanomedicine in the drug development process.
Assuntos
Neoplasias Colorretais , Polinucleotídeo 5'-Hidroxiquinase , Animais , Neoplasias Colorretais/tratamento farmacológico , Camundongos , Camundongos Nus , Nanomedicina , PTEN Fosfo-Hidrolase/deficiência , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidoresRESUMO
The structure-specific ERCC1-XPF endonuclease is essential for repairing bulky DNA lesions and helix distortions induced by UV radiation, which forms cyclobutane pyrimidine dimers (CPDs), or chemicals that crosslink DNA strands such as cyclophosphamide and platinum-based chemotherapeutic agents. Inhibition of the ERCC1-XPF endonuclease activity has been shown to sensitize cancer cells to these chemotherapeutic agents. In this study, we have conducted a structure activity relationship analysis based around the previously identified hit compound, 4-((6-chloro-2-methoxyacridin-9-yl)amino)-2-((4-methylpiperazin1-yl)methyl)phenol (F06), as a reference compound. Three different series of compounds have been rationally designed and successfully synthesized through various modifications on three different sites of F06 based on the corresponding suggestions of the previous pharmacophore model. The in vitro screening results revealed that 2-chloro-9-((3-((4-(2-(dimethylamino)ethyl)piperazin-1-yl)methyl)-4-hydroxyphenyl)amino)acridin-2-ol (B9) has a potent inhibitory effect on the ERCC1-XPF activity (IC50 = 0.49 µM), showing 3-fold improvement in inhibition activity compared to F06. In addition, B9 not only displayed better binding affinity to the ERCC1-XPF complex but also had the capacity to potentiate the cytotoxicity effect of UV radiation and inhibiting the nucleotide excision repair, by the inhibition of removal of CPDs, and cyclophosphamide toxicity to colorectal cancer cells.
Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/antagonistas & inibidores , Desenho de Fármacos , Endonucleases/antagonistas & inibidores , Linhagem Celular Tumoral , Sistema Livre de Células , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Humanos , Técnicas In VitroRESUMO
The ERCC1-XPF heterodimer is a 5'-3' structure-specific endonuclease, which plays an essential role in several DNA repair pathways in mammalian cells. ERCC1-XPF is primarily involved in the repair of chemically induced helix-distorting and bulky DNA lesions, such as cyclobutane pyrimidine dimers (CPDs), and DNA interstrand cross-links. Inhibition of ERCC1-XPF has been shown to potentiate cytotoxicity of platinum-based drugs and cyclophosphamide in cancer cells. In this study, the previously described ERCC1-XPF inhibitor 4-((6-chloro-2-methoxyacridin-9-yl)amino)-2-((4-methylpiperazin-1-yl)methyl)phenol (compound 1) was used as a reference compound. Following the outcome of docking-based virtual screening (VS), we synthesized seven novel derivatives of 1 that were identified in silico as being likely to have high binding affinity for the ERCC1-XPF heterodimerization interface by interacting with the XPF double helix-hairpin-helix (HhH2) domain. Two of the new compounds, 4-((6-chloro-2-methoxyacridin-9-yl)amino)-2-((4-cyclohexylpiperazin-1-yl)methyl)phenol (compound 3) and 4-((6-chloro-2-methoxyacridin-9-yl)amino)-2-((4-(2-(dimethylamino)ethyl) piperazin-1-yl) methyl) phenol (compound 4), were shown to be potent inhibitors of ERCC1-XPF activity in vitro. Compound 4 showed significant inhibition of the removal of CPDs in UV-irradiated cells and the capacity to sensitize colorectal cancer cells to UV radiation and cyclophosphamide.
Assuntos
Reparo do DNA , DNA de Neoplasias/efeitos dos fármacos , Proteínas de Ligação a DNA/antagonistas & inibidores , Endonucleases/antagonistas & inibidores , Pirimidinas/farmacologia , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Desenho de Fármacos , Endonucleases/metabolismo , Células HCT116 , Humanos , Modelos Moleculares , Estrutura Molecular , Pirimidinas/síntese química , Pirimidinas/química , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Deoxycytidine kinase (dCK), a cytosolic enzyme with broad substrate specificity, plays a key role in the activation of therapeutic nucleoside analogues by their 5'-phosphorylation. The structure of human dCK is still not known and the current work was undertaken to determine its oligomeric and secondary structure. Biophysical studies were conducted with purified recombinant human dCK. The Mr determined by low-speed sedimentation equilibrium under nondenaturing conditions was 60,250 +/- 1,000, indicating that dCK, which has a predicted Mr of 30,500, exists in solution as a dimer. Analysis of circular dichroism spectra revealed the presence of two negative dichroic bands located at 222 and 209 nm with ellipticity values of -11,900 +/- 300 and -12,500 +/- 300 deg x cm2 x dmol(-1), respectively, indicating the presence of approximately 40% alpha-helix and 50% beta-structure. Circular Dichroism studies in the aromatic and far-ultraviolet range and UV difference spectroscopy indicated that binding of substrates to dCK reduced its alpha-helical content and perturbed tryptophan and tyrosine. Steady-state fluorescence demonstrated that deoxycytidine (the phosphate acceptor) and ATP (the phosphate donor) bound to different sites on dCK and fluorescence quenching revealed bimodal binding of deoxycytidine and unimodal binding of ATP. Spectroscopic studies indicated that substrate binding induced conformational changes, with the result that dCK exhibited different affinities for various substrates. These results are consistent with a random bi-bi kinetic mechanism of phosphorylation of dCyd with either ATP or UTP.
Assuntos
Desoxicitidina Quinase/química , Desoxicitidina Quinase/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Cromatografia em Gel , Dicroísmo Circular , DNA Complementar/química , Desoxicitidina/análogos & derivados , Desoxicitidina/metabolismo , Dimerização , Humanos , Ligantes , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrofotometria Ultravioleta , Especificidade por Substrato , Ultracentrifugação , Uridina Trifosfato/química , Uridina Trifosfato/metabolismoRESUMO
Human polynucleotide kinase (hPNK) is required for processing and rejoining DNA strand break termini. The 5'-DNA kinase and 3'-phosphatase activities of hPNK can be stimulated by the "scaffold" protein XRCC1, but the mechanism remains to be fully elucidated. Using a variety of fluorescence techniques, we examined the interaction of hPNK with XRCC1 and substrates that model DNA single-strand breaks. hPNK binding to substrates with 5'-OH termini was only approximately 5-fold tighter than that to identical DNA molecules with 5'-phosphate termini, suggesting that hPNK remains bound to the product of its enzymatic activity. The presence of XRCC1 did not influence the binding of hPNK to substrates with 5'-OH termini, but sharply reduced the interaction of hPNK with DNA bearing a 5'-phosphate terminus. These data, together with kinetic data obtained at limiting enzyme concentration, indicate a dual function for the interaction of XRCC1 with hPNK. First, XRCC1 enhances the capacity of hPNK to discriminate between strand breaks with 5'-OH termini and those with 5'-phosphate termini; and second, XRCC1 stimulates hPNK activity by displacing hPNK from the phosphorylated DNA product.
Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/fisiologia , Polinucleotídeo 5'-Hidroxiquinase/metabolismo , 2-Naftilamina/análogos & derivados , 2-Naftilamina/química , Sequência de Bases , Dano ao DNA , Transferência Ressonante de Energia de Fluorescência , Humanos , Cinética , Modelos Biológicos , Conformação Molecular , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Especificidade por Substrato , Proteína 1 Complementadora Cruzada de Reparo de Raio-XRESUMO
Human deoxycytidine kinase (dCK) phosphorylates both pyrimidine and purine deoxynucleosides, including numerous nucleoside analogue prodrugs. Energy transfer studies of transfer between Trp residues of dCK and the fluorescent probe N-(1-pyrene)maleimide (PM), which specifically labels Cys residues in proteins, were performed. Two of the six Cys residues in dCK were labeled, yielding a protein that was functionally active. We determined the average distances between PM-labeled Cys residues and Trp residues in dCK in the absence and presence of various pyrimidine and purine nucleoside analogues with the Trp residues as energy donors and PM-labeled Cys residues as acceptors. The transfer efficiency was determined from donor intensity quenching and the Förster distance R(0) at which the efficiency of energy transfer is 50%, which was 19.90 A for dCK-PM. The average distance R between the Trp residues and the labeled Cys residues in dCK-PM was 18.50 A, and once substrates bound, this distance was reduced, demonstrating conformational changes. Several of the Cys residues of dCK were mutated to Ala, and the properties of the purified mutant proteins were studied. PM labeled a single Cys residue in Cys-185-Ala dCK, suggesting that one of the two Cys residues labeled in wild-type dCK was Cys 185. The distance between the single PM-labeled Cys residue and the Trp residues in Cys-185-Ala dCK was 20.75 A. Binding of nucleosides had no effect on the pyrene fluorescence of Cys-185-Ala dCK, indicating that the conformational changes observed upon substrate binding to wild-type dCK-PM involved the "lid region" of which Cys 185 is a part. The substrate specificity of Cys-185-Ala dCK was altered in that dAdo and UTP were better substrates for the mutant than for the wild-type enzyme.
Assuntos
Cisteína/metabolismo , Desoxicitidina Quinase/química , Transferência Ressonante de Energia de Fluorescência/métodos , Conformação Proteica , Alanina/química , Alanina/genética , Alanina/metabolismo , Sítios de Ligação , Cisteína/química , Cisteína/genética , Desoxicitidina Quinase/genética , Desoxicitidina Quinase/metabolismo , Transferência de Energia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Corantes Fluorescentes/metabolismo , Corantes Fluorescentes/farmacologia , Humanos , Cinética , Maleimidas/metabolismo , Maleimidas/farmacologia , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Nucleosídeos/metabolismo , Nucleosídeos/farmacologia , Fosfatos/metabolismo , Ligação Proteica , Especificidade por Substrato/efeitos dos fármacos , Especificidade por Substrato/genética , Triptofano/químicaRESUMO
Mammalian polynucleotide kinase (PNK) is a key component of both the base excision repair (BER) and nonhomologous end-joining (NHEJ) DNA repair pathways. PNK acts as a 5'-kinase/3'-phosphatase to create 5'-phosphate/3'-hydroxyl termini, which are a necessary prerequisite for ligation during repair. PNK is recruited to repair complexes through interactions between its N-terminal FHA domain and phosphorylated components of either pathway. Here, we describe the crystal structure of intact mammalian PNK and a structure of the PNK FHA bound to a cognate phosphopeptide. The kinase domain has a broad substrate binding pocket, which preferentially recognizes double-stranded substrates with recessed 5' termini. In contrast, the phosphatase domain efficiently dephosphorylates single-stranded 3'-phospho termini as well as double-stranded substrates. The FHA domain is linked to the kinase/phosphatase catalytic domain by a flexible tether, and it exhibits a mode of target selection based on electrostatic complementarity between the binding surface and the phosphothreonine peptide.
Assuntos
Reparo do DNA , Polinucleotídeo 5'-Hidroxiquinase/fisiologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Glutationa Transferase/metabolismo , Cinética , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Oligonucleotídeos/química , Monoéster Fosfórico Hidrolases/química , Fosforilação , Polinucleotídeo 5'-Hidroxiquinase/química , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Espectrometria de Fluorescência , Eletricidade Estática , Especificidade por Substrato , Tripsina/químicaRESUMO
Human polynucleotide kinase (hPNK), which possesses both 5'-DNA kinase and 3'-DNA phosphatase activities, is a DNA repair enzyme required for processing and rejoining of single- and double-strand-break termini. Full-length hPNK was subjected to sedimentation and spectroscopic analyses in association with its ligands, a 20-mer oligonucleotide, ATP, and AMP-PNP (a nonhydrolyzable analogue of ATP). Sedimentation equilibrium measurements indicated that hPNK was a monomer in the presence and absence of the ligands. Circular dichroism measurements revealed that the ligands induced different conformational changes in hPNK, although AMP-PNP induced the same conformational changes as ATP. CD also indicated that the oligonucleotide could bind to the protein-AMP-PNP complex. Protein-ligand binding affinities and stoichiometries were determined by measuring changes in protein intrinsic fluorescence. Titrating hPNK with the oligonucleotide indicated tight binding with a K(d) value of 1.3 microM and with 1:1 stoichiometry. A 5'-phosphorylated oligonucleotide with the same sequence exhibited an almost 6-fold lower affinity (K(d) value, 7.2 microM). ATP and AMP-PNP bound with high affinity (K(d) values, respectively, of 1.4 and 1.6 microM), and the observed binding stoichiometries were 1:1. Furthermore, the nonphosphorylated oligonucleotide was able to bind to hPNK in the presence of AMP-PNP with a K(d) value of 2.5 microM, confirming the formation of a ternary complex. This study provides the first direct physical evidence for such a ternary complex involving a polynucleotide kinase, AMP-PNP, and an oligonucleotide, and supports a reaction mechanism in which ATP and DNA bind simultaneously to the enzyme.
Assuntos
Trifosfato de Adenosina/química , Adenilil Imidodifosfato/química , Proteínas de Ligação a DNA/química , Oligonucleotídeos/química , Polinucleotídeo 5'-Hidroxiquinase/química , Sítios de Ligação , Dicroísmo Circular , Humanos , Ligantes , Fosforilação , Polinucleotídeo 5'-Hidroxiquinase/genética , Polinucleotídeo 5'-Hidroxiquinase/isolamento & purificação , Conformação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Espectrometria de Fluorescência , UltracentrifugaçãoRESUMO
The human DNA repair protein, hXRCC1, which is required for DNA single-strand break repair and genetic stability was produced as a histidine-tagged polypeptide in Escherichia coli, purified by affinity chromatography, and subjected to sedimentation and spectroscopic analyses. This study represents the first biophysical examination of full-length XRCC1. Sedimentation equilibrium measurements indicated that hXRCC1 exists as a monomer at lower protein concentrations but forms a dimer at higher protein concentrations with a K(d) of 5.7 x 10(-)(7) M. The size and shape of hXRCC1 in solution were determined by analytical ultracentrifugation studies. The protein exhibited an intrinsic sedimentation coefficient, s(0)(20,w), of 3.56 S and a Stokes radius, R(s), of 44.5 A, which together with the M(r) of 68000 suggested that hXRCC1 is a moderately asymmetric protein with an axial ratio of 7.2. Binding of model ligands, representing single-strand breaks with either a nick or a single nucleotide gap, quenched protein fluorescence, and binding affinities and stoichiometries were determined by carrying out fluorescence titrations as a function of ligand concentration. XRCC1 bound both nicked and 1 nucleotide-gapped DNA substrates tightly in a stoichiometric manner (1:1) with K(d) values of 65 and 34 nM, respectively. However, hXRCC1 exhibited lower affinities for a duplex with a 5 nucleotide gap, the intact duplex with no break, and a single-stranded oligonucleotide with K(d) values of 215, 230, and 260 nM, respectively. Our results suggest that hXRCC1 exhibits preferential binding to DNA with single-strand breaks with a gap size of <5 nucleotides.