RESUMEN
Theta-mediated end joining (TMEJ) is critical for survival of cancer cells when other DNA double-stranded break repair pathways are impaired. Human DNA polymerase theta (Pol θ) can extend ssDNA oligonucleotides, but little is known about preferred substrates and mechanism. We show that Pol θ can extend both ssDNA and RNA substrates by unimolecular stem-loop synthesis initiated by only two 3' terminal base pairs. Given sufficient time, Pol θ uses alternative pairing configurations that greatly expand the repertoire of sequence outcomes. Further primer-template adjustments yield low-fidelity outcomes when the nucleotide pool is imbalanced. Unimolecular stem-loop synthesis competes with bimolecular end joining, even when a longer terminal microhomology for end joining is available. Both reactions are partially suppressed by the ssDNA-binding protein replication protein A. Protein-primer grasp residues that are specific to Pol θ are needed for rapid stem-loop synthesis. The ability to perform stem-loop synthesis from a minimally paired primer is rare among human DNA polymerases, but we show that human DNA polymerases Pol η and Pol λ can catalyze related reactions. Using purified human Pol θ, we reconstituted in vitro TMEJ incorporating an insertion arising from a stem-loop extension. These activities may help explain TMEJ repair events that include inverted repeat sequences.
Asunto(s)
ADN Polimerasa theta , ADN Polimerasa Dirigida por ADN , Humanos , ADN Polimerasa Dirigida por ADN/metabolismo , ADN Polimerasa Dirigida por ADN/genética , ADN de Cadena Simple/metabolismo , ADN de Cadena Simple/genética , ADN de Cadena Simple/química , Reparación del ADN por Unión de Extremidades , Reparación del ADN , Proteína de Replicación A/metabolismo , Proteína de Replicación A/genética , ADN Polimerasa beta/metabolismo , ADN Polimerasa beta/genética , ADN Polimerasa beta/químicaRESUMEN
Genogroup II (GII) noroviruses are a major cause of diarrheal disease burden in children in both high- and low-income countries. GII.17 noroviruses are composed of distinct genetic clusters (I, II, IIIa, and IIIb) and have shown potential for replacing historically more prevalent GII.4 strains, but the serological basis for GII.17 antigenic diversity has not been studied in children. Utilizing samples from a birth cohort, we investigated antibody and B-cell responses to GII.17 cluster variants in confirmed GII.17 infections in young children as well as demonstrated that the distinct genetic clusters co-circulate. Polyclonal serum antibodies bound multiple clusters but showed cluster-specific blockade activity in a surrogate virus neutralization assay. Antibodies secreted by immortalized memory B cells (MBCs) from an infant GII.17 case were highly specific to GII.17 and exhibited blockade activity against this genotype. We isolated an MBC-derived GII.17-specific Immunoglobulin A (IgA) monoclonal antibody called NVA.1 that potently and selectively blocked GII.17 cluster IIIb and recognized an epitope targeted in serum from cluster IIIb-infected children. These data indicate that multiple antigenically distinct GII.17 variants co-circulate in young children, suggesting retention of cluster diversity alongside potential for immune escape given the existence of antibody-defined cluster-specific epitopes elicited during infection.
Asunto(s)
Linfocitos B , Norovirus , Niño , Lactante , Humanos , Preescolar , Anticuerpos Monoclonales , Células B de Memoria , Inmunoglobulina A , Paraproteínas , Epítopos , Genotipo , Norovirus/genéticaRESUMEN
Oxidative damage on DNA arising from both endogenous and exogenous sources can result in base modifications that promote errors in replication as well as generating sites of base loss (abasic sites) that present unique challenges to maintaining genomic integrity. These lesions are excised by DNA glycosylases in the first step of the base excision repair pathway. Here we present the first crystal structure of a NEIL2 glycosylase, an enzyme active on cytosine oxidation products and abasic sites. The structure reveals an unusual "open" conformation not seen in NEIL1 or NEIL3 orthologs. NEIL2 is predicted to adopt a "closed" conformation when bound to its substrate. Combined crystallographic and solution-scattering studies show the enzyme to be conformationally dynamic in a manner distinct among the NEIL glycosylases and provide insight into the unique substrate preference of this enzyme. In addition, we characterized three cancer variants of human NEIL2, namely S140N, G230W, and G303R.
Asunto(s)
ADN Glicosilasas/química , ADN-(Sitio Apurínico o Apirimidínico) Liasa/química , Sitios de Unión , ADN/química , ADN/metabolismo , ADN Glicosilasas/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Humanos , Simulación de Dinámica Molecular , Unión ProteicaRESUMEN
MutY glycosylase excises adenines misincorporated opposite the oxidatively damaged lesion, 8-oxo-7,8-dihydroguanine (OG), to initiate base excision repair and prevent G to T transversion mutations. Successful repair requires MutY recognition of the OG:A mispair amidst highly abundant and structurally similar undamaged DNA base pairs. Herein we use a combination of in vitro and bacterial cell repair assays with single-molecule fluorescence microscopy to demonstrate that both a C-terminal domain histidine residue and the 2-amino group of OG base are critical for MutY detection of OG:A sites. These studies are the first to directly link deficiencies in MutY lesion detection with incomplete cellular repair. These results suggest that defects in lesion detection of human MutY (MUTYH) variants may prove predictive of early-onset colorectal cancer known an MUTYH-associated polyposis. Furthermore, unveiling these specific molecular determinants for repair makes it possible to envision new MUTYH-specific cancer therapies.
Asunto(s)
ADN Glicosilasas/metabolismo , Guanina/análogos & derivados , Histidina/metabolismo , ADN Glicosilasas/química , Guanina/análisis , Guanina/metabolismo , Humanos , Microscopía Fluorescente , Modelos MolecularesRESUMEN
The chimeric DnaJ-PKAc enzymeresulting from an approximately 400-kb deletion of chromosome 19 is a primary contributor to the oncogenic transformation that occurs in fibrolamellar hepatocellular carcinoma, also called fibrolamellar carcinoma (FLC). This oncogenic deletion juxtaposes exon 1 of the DNAJB1 heat shock protein gene with exon 2 of the PRKACA gene encoding the protein kinase A catalytic subunit, resulting in DnaJ-PKAc fusion under the transcriptional control of the DNAJB1 promoter. The expression of DnaJ-PKAc is approximately 10 times that of wild-type (wt) PKAc catalytic subunits, causing elevated and dysregulated kinase activity that contributes to oncogenic transformation. In normal cells, PKAc activity is regulated by a group of endogenous proteins, termed protein kinase inhibitors (PKI) that competitively inhibit PKAc and assist with the nuclear export of the enzyme. Currently, it is scarcely known whether interactions with PKI are perturbed in DnaJ-PKAc. In this report, we survey existing data sets to assess the expression levels of the various PKI isoforms that exist in humans to identify those that are candidates to encounter DnaJ-PKAc in both normal liver and FLC tumors. We then compare inhibition profiles of wtPKAc and DnaJ-PKAc against PKI and demonstrate that extensive structural homology in the active site clefts of the two enzymes confers similar kinase activities and inhibition by full-length PKI and PKI-derived peptides.
Asunto(s)
Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico , Proteínas del Choque Térmico HSP40 , Proteínas de Fusión Oncogénica , Péptidos/química , Inhibidores de Proteínas Quinasas/química , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/antagonistas & inhibidores , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/química , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/genética , Proteínas del Choque Térmico HSP40/antagonistas & inhibidores , Proteínas del Choque Térmico HSP40/química , Proteínas del Choque Térmico HSP40/genética , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Proteínas de Fusión Oncogénica/antagonistas & inhibidores , Proteínas de Fusión Oncogénica/química , Proteínas de Fusión Oncogénica/genéticaRESUMEN
Proper repair of oxidatively damaged DNA bases is essential to maintain genome stability. 8-Oxoguanine (7,8-dihydro-8-oxoguanine, 8-oxoG) is a dangerous DNA lesion because it can mispair with adenine (A) during replication resulting in guanine to thymine transversion mutations. MUTYH DNA glycosylase is responsible for recognizing and removing the adenine from 8-oxoG:adenine (8-oxoG:A) sites. Biallelic mutations in the MUTYH gene predispose individuals to MUTYH-associated polyposis (MAP), and the most commonly observed mutation in some MAP populations is Y165C. Tyr165 is a 'wedge' residue that intercalates into the DNA duplex in the lesion bound state. Here, we utilize single molecule fluorescence microscopy to visualize the real-time search behavior of Escherichia coli and Mus musculus MUTYH WT and wedge variant orthologs on DNA tightropes that contain 8-oxoG:A, 8-oxoG:cytosine, or apurinic product analog sites. We observe that MUTYH WT is able to efficiently find 8-oxoG:A damage and form highly stable bound complexes. In contrast, MUTYH Y150C shows decreased binding lifetimes on undamaged DNA and fails to form a stable lesion recognition complex at damage sites. These findings suggest that MUTYH does not rely upon the wedge residue for damage site recognition, but this residue stabilizes the lesion recognition complex.
Asunto(s)
Poliposis Adenomatosa del Colon/genética , Neoplasias Colorrectales/genética , Daño del ADN/genética , ADN Glicosilasas/genética , Adenina/metabolismo , Poliposis Adenomatosa del Colon/patología , Animales , Neoplasias Colorrectales/patología , Escherichia coli/genética , Inestabilidad Genómica/genética , Guanina/análogos & derivados , Guanina/química , Humanos , Ratones , Mutación , Estrés Oxidativo/genéticaRESUMEN
The human RAD51 recombinase possesses DNA pairing and strand exchange activities that are essential for the error-free, homology-directed repair of DNA double-strand breaks. The recombination activities of RAD51 are activated upon its assembly into presynaptic filaments on single-stranded DNA at resected DSB ends. Defects in filament assembly caused by mutations in RAD51 or its regulators such as BRCA2 are associated with human cancer. Here we describe two novel RAD51 missense variants located in the multimerization/BRCA2 binding region of RAD51. F86L is a breast tumor-derived somatic variant that affects the interface between adjacent RAD51 protomers in the presynaptic filament. E258A is a germline variant that maps to the interface region between the N-terminal and RecA homology domains of RAD51. Both variants exhibit abnormal biochemistry including altered DNA strand exchange activity. Both variants inhibit the DNA strand exchange activity of wild-type RAD51, suggesting a mechanism for negative dominance. The inhibitory effect of F86L on wild-type RAD51 is surprising since F86L alone exhibits robust DNA strand exchange activity. Our findings indicate that even DNA strand exchange-proficient variants can have negative functional interactions with wild-type RAD51. Thus heterozygous F86L or E258 mutations in RAD51 could promote genomic instability, and thereby contribute to tumor progression.
Asunto(s)
Proteína BRCA2/metabolismo , Mutación Missense , Dominios y Motivos de Interacción de Proteínas , Recombinasa Rad51/metabolismo , Reparación del ADN por Recombinación , Secuencia de Aminoácidos , ADN/metabolismo , Roturas del ADN de Doble Cadena , Predisposición Genética a la Enfermedad , Humanos , Cinética , Modelos Moleculares , Neoplasias/genética , Neoplasias/metabolismo , Unión Proteica , Multimerización de Proteína , Recombinasa Rad51/química , Recombinasa Rad51/genética , Alineación de SecuenciaRESUMEN
DNA polymerase θ (Pol θ) is implicated in various cellular processes including double-strand break repair and apurinic/apyrimidinic site bypass. Because Pol θ expression correlates with poor cancer prognosis, the ability of Pol θ to bypass the C4'-oxidized abasic site (C4-AP) and 2-deoxyribonolactone (L), which are generated by cytotoxic agents, is of interest. Translesion synthesis and subsequent extension by Pol θ past C4-AP or L and an abasic site (AP) or its tetrahydrofuran analogue (F) was examined. Pol θ conducts translesion synthesis on templates containing AP and F with similar efficiencies and follows the "A-rule," inserting nucleotides in the order A > G > T. Translesion synthesis on templates containing C4-AP and L is less efficient than AP and F, and the preference for A insertion is reduced for L and absent for C4-AP. Extension past all abasic lesions (AP, F, C4-AP, and L) was significantly less efficient than translesion synthesis and yielded deletions caused by the base one or two nucleotides downstream from the lesion being used as a template, with the latter being favored. These results suggest that bypass of abasic lesions by Pol θ is highly mutagenic.
Asunto(s)
ADN Polimerasa beta/fisiología , Mutagénesis , Animales , Secuencia de Bases , Daño del ADN , ADN Polimerasa beta/metabolismo , Reparación del ADN , Humanos , Nucleótidos/metabolismo , Oxidación-Reducción , Eliminación de Secuencia , Azúcares Ácidos , Moldes GenéticosRESUMEN
In human cells, error-free repair of DNA double-strand breaks requires the DNA pairing and strand exchange activities of RAD51 recombinase. Activation of RAD51 recombination activities requires the assembly of RAD51 presynaptic filaments on the single-stranded DNA that forms at resected DSB ends. Mutations in proteins that control presynaptic filament assembly, such as BRCA2, and in RAD51 itself, are associated with human breast cancer. Here we describe the properties of two mutations in RAD51 protein that derive from human lung and kidney tumors, respectively. Sequence variants Q268P and Q272L both map to the DNA binding loop 2 (L2) region of RAD51, a motif that is involved in DNA binding and in the allosteric activation of ATP hydrolysis and DNA strand exchange activities. Both mutations alter the thermal stability, DNA binding, and ATPase properties of RAD51, however both variants retain intrinsic DNA strand exchange activity towards oligonucleotide substrates under optimized conditions. In contrast, both Q268P and Q272L variants exhibit drastically reduced DNA strand exchange activity in reaction mixtures containing long homologous ssDNA and dsDNA substrates and human RPA protein. Mixtures of wild-type and variant proteins also exhibit reduced DNA strand exchange activity, suggesting that heterozygous mutations could negatively affect DNA recombination and repair processes in vivo. Together, the findings of this study suggest that hypomorphic missense mutations in RAD51 protein could be drivers of genomic instability in cancer cells, and thereby contribute to the etiology of metastatic disease.
Asunto(s)
ADN/genética , Neoplasias Renales/genética , Neoplasias Renales/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Mutación Missense , Recombinasa Rad51/genética , ADN/metabolismo , Femenino , Humanos , Persona de Mediana Edad , Modelos Moleculares , Conformación Proteica , Estabilidad Proteica , Recombinasa Rad51/química , Recombinasa Rad51/metabolismo , TemperaturaRESUMEN
DNA polymerase θ protects against genomic instability via an alternative end-joining repair pathway for DNA double-strand breaks. Polymerase θ is overexpressed in breast, lung and oral cancers, and reduction of its activity in mammalian cells increases sensitivity to double-strand break-inducing agents, including ionizing radiation. Reported here are crystal structures of the C-terminal polymerase domain from human polymerase θ, illustrating two potential modes of dimerization. One structure depicts insertion of ddATP opposite an abasic-site analog during translesion DNA synthesis. The second structure describes a cognate ddGTP complex. Polymerase θ uses a specialized thumb subdomain to establish unique upstream contacts to the primer DNA strand, including an interaction with the 3'-terminal phosphate from one of five distinctive insertion loops. These observations demonstrate how polymerase θ grasps the primer to bypass DNA lesions or extend poorly annealed DNA termini to mediate end-joining.
Asunto(s)
ADN Polimerasa Dirigida por ADN/fisiología , Secuencia de Aminoácidos , Cristalografía por Rayos X , ADN Polimerasa Dirigida por ADN/química , Dimerización , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Alineación de Secuencia , ADN Polimerasa thetaRESUMEN
G-quadruplex is a four-stranded G-rich DNA structure that is highly susceptible to oxidation. Despite the important roles that G-quadruplexes play in telomere biology and gene transcription, neither the impact of guanine lesions on the stability of quadruplexes nor their repair are well understood. Here, we show that the oxidized guanine lesions 8-oxo-7,8-dihydroguanine (8-oxoG), guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp) reduce the thermostability and alter the folding of telomeric quadruplexes in a location-dependent manner. Also, the NEIL1 and NEIL3 DNA glycosylases can remove hydantoin lesions but none of the glycosylases, including OGG1, are able to remove 8-oxoG from telomeric quadruplexes. Interestingly, a hydantoin lesion at the site most prone to oxidation in quadruplex DNA is not efficiently removed by NEIL1 or NEIL3. However, NEIL1, NEIL2 and NEIL3 remove hydantoins from telomeric quadruplexes formed by five TTAGGG repeats much more rapidly than the commonly studied four-repeat quadruplex structures. We also show that APE1 cleaves furan in selected positions in Na(+)-coordinated telomeric quadruplexes. In promoter G-quadruplex DNA, the NEIL glycosylases primarily remove Gh from Na(+)-coordinated antiparallel quadruplexes but not K(+)-coordinated parallel quadruplexes containing VEGF or c-MYC promoter sequences. Thus, the NEIL DNA glycosylases may be involved in both telomere maintenance and in gene regulation.
Asunto(s)
ADN Glicosilasas/metabolismo , G-Cuádruplex , Guanina/metabolismo , N-Glicosil Hidrolasas/metabolismo , Regiones Promotoras Genéticas , Telómero/metabolismo , ADN/química , ADN/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Furanos/metabolismo , Guanidinas/metabolismo , Guanina/análogos & derivados , Guanosina/análogos & derivados , Guanosina/metabolismo , Humanos , Hidantoínas/metabolismo , Oxidación-Reducción , Potasio/química , Sodio/química , Compuestos de Espiro/metabolismo , Telómero/químicaRESUMEN
Human RAD51 protein catalyzes DNA pairing and strand exchange reactions that are central to homologous recombination and homology-directed DNA repair. Successful recombination/repair requires the formation of a presynaptic filament of RAD51 on ssDNA. Mutations in BRCA2 and other proteins that control RAD51 activity are associated with human cancer. Here we describe a set of mutations associated with human breast tumors that occur in a common structural motif of RAD51. Tumor-associated D149N, R150Q and G151D mutations map to a Schellman loop motif located on the surface of the RecA homology domain of RAD51. All three variants are proficient in DNA strand exchange, but G151D is slightly more sensitive to salt than wild-type (WT). Both G151D and R150Q exhibit markedly lower catalytic efficiency for adenosine triphosphate hydrolysis compared to WT. All three mutations alter the physical properties of RAD51 nucleoprotein filaments, with G151D showing the most dramatic changes. G151D forms mixed nucleoprotein filaments with WT RAD51 that have intermediate properties compared to unmixed filaments. These findings raise the possibility that mutations in RAD51 itself may contribute to genome instability in tumor cells, either directly through changes in recombinase properties, or indirectly through changes in interactions with regulatory proteins.
Asunto(s)
Mutación , Neoplasias/genética , Recombinasa Rad51/química , Recombinasa Rad51/genética , Adenosina Trifosfato/metabolismo , Secuencias de Aminoácidos/genética , ADN/metabolismo , Humanos , Modelos Moleculares , Pliegue de Proteína , Recombinasa Rad51/metabolismoRESUMEN
Assault to DNA that leads to oxidative base damage is repaired by the base excision repair (BER) pathway with specialized enzymes called DNA glycosylases catalyzing the first step of this pathway. These glycosylases can be categorized into two families: the HhH superfamily, which includes endonuclease III (or Nth), and the Fpg/Nei family, which comprises formamidopyrimidine DNA glycosylase (or Fpg) and endonuclease VIII (or Nei). In humans there are three Nei-like (NEIL) glycosylases: NEIL1, 2, and 3. Here we present the first crystal structure of a viral ortholog of the human NEIL2/NEIL3 proteins, Mimivirus Nei2 (MvNei2), determined at 2.04Å resolution. The C-terminal region of the MvNei2 enzyme comprises two conserved DNA binding motifs: the helix-two-turns-helix (H2TH) motif and a C-H-C-C type zinc-finger similar to that of human NEIL2. The N-terminal region of MvNei2 is most closely related to NEIL3. Like NEIL3, MvNei2 bears a valine at position 2 instead of the usual proline and it lacks two of the three conserved void-filling residues present in other members of the Fpg/Nei family. Mutational analysis of the only conserved void-filling residue methionine 72 to alanine yields an MvNei2 variant with impaired glycosylase activity. Mutation of the adjacent His73 causes the enzyme to be more productive thereby suggesting a plausible role for this residue in the DNA lesion search process.
Asunto(s)
ADN Glicosilasas/química , ADN Glicosilasas/metabolismo , Mimiviridae/enzimología , Proteínas Virales/química , Proteínas Virales/metabolismo , Dedos de Zinc , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Clonación Molecular , Secuencia de Consenso , Cristalografía por Rayos X , Daño del ADN/genética , ADN Glicosilasas/genética , Reparación del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa/química , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Humanos , Mimiviridae/genética , Mutagénesis Sitio-Dirigida , N-Glicosil Hidrolasas/química , N-Glicosil Hidrolasas/genética , N-Glicosil Hidrolasas/metabolismo , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia , Proteínas Virales/genéticaRESUMEN
Endonuclease VIII-like 3 (Neil3) is a DNA glycosylase of the base excision repair pathway that protects cells from oxidative DNA damage by excising a broad spectrum of cytotoxic and mutagenic base lesions. Interestingly, Neil3 exhibits an unusual preference for DNA with single-stranded regions. Here, we report the 2.0 Å crystal structure of a Neil3 enzyme. Although the glycosylase region of mouse Neil3 (MmuNeil3Δ324) exhibits the same overall fold as that of other Fpg/Nei proteins, it presents distinct structural features. First, MmuNeil3Δ324 lacks the αF-ß9/10 loop that caps the flipped-out 8-oxoG in bacterial Fpg, which is consistent with its inability to cleave 8-oxoguanine. Second, Neil3 not only lacks two of the three void-filling residues that stabilize the opposite strand, but it also harbors negatively charged residues that create an unfavorable electrostatic environment for the phosphate backbone of that strand. These structural features provide insight into the substrate specificity and marked preference of Neil3 for ssDNA.
Asunto(s)
ADN de Cadena Simple/química , Endodesoxirribonucleasas/química , Secuencias de Aminoácidos , Animales , Dominio Catalítico , Cristalografía por Rayos X , Humanos , Enlace de Hidrógeno , Ratones , Modelos Moleculares , Conformación de Ácido Nucleico , Unión Proteica , Estabilidad Proteica , Estructura Secundaria de Proteína , Especificidad por Sustrato , Propiedades de SuperficieRESUMEN
Endonuclease VIII-like 3 (Neil3) is one of the five DNA glycosylases found in mammals that recognize and remove oxidized bases, and initiate the base excision repair (BER) pathway. Previous attempts to express and purify the mouse and human orthologs of Neil3 in their active form have not been successful. Here we report the construction of bicistronic expression vectors for expressing in Escherichia coli the full-length mouse Neil3 (MmuNeil3), its glycosylase domain (MmuNeil3Δ324), as well as the glycosylase domain of human Neil3 (NEIL3Δ324). The purified Neil3 proteins are all active, and NEIL3Δ324 exhibits similar glycosylase/lyase activity as MmuNeil3Δ324 on both single-stranded and double-stranded substrates containing thymine glycol (Tg), spiroiminodihydantoin (Sp) or an abasic site (AP). We show that N-terminal initiator methionine processing is critical for the activity of both mouse and human Neil3 proteins. Co-expressing an E. coli methionine aminopeptidase (EcoMap) Y168A variant with MmuNeil3, MmuNeil3Δ324 and NEIL3Δ324 improves the N-terminal methionine processing and increases the percentage of active Neil3 proteins in the preparation. The purified Neil3 proteins are suitable for biochemical, structural and functional studies.
Asunto(s)
Endodesoxirribonucleasas/biosíntesis , Endodesoxirribonucleasas/aislamiento & purificación , N-Glicosil Hidrolasas/biosíntesis , N-Glicosil Hidrolasas/aislamiento & purificación , Proteínas Recombinantes de Fusión/aislamiento & purificación , Animales , Clonación Molecular , Codón , Electroforesis en Gel de Poliacrilamida , Endodesoxirribonucleasas/química , Endodesoxirribonucleasas/genética , Estabilidad de Enzimas , Escherichia coli/genética , Escherichia coli/metabolismo , Histidina/química , Histidina/genética , Histidina/metabolismo , Humanos , Metionina/metabolismo , Ratones , N-Glicosil Hidrolasas/química , N-Glicosil Hidrolasas/genética , Oligopéptidos/química , Oligopéptidos/genética , Oligopéptidos/metabolismo , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Thymine glycol (Tg) and 5-hydroxyuracil (5-OHU) are common oxidized products of pyrimidines, which are recognized and cleaved by two DNA glycosylases of the base excision repair pathway, endonuclease III (Nth) and endonuclease VIII (Nei). Although there are several structures of Nei enzymes unliganded or bound to an abasic (apurinic or apyrimidinic) site, until now there was no structure of an Nei bound to a DNA lesion. Mimivirus Nei1 (MvNei1) is an ortholog of human NEIL1, which was previously crystallized bound to DNA containing an apurinic site (Imamura, K., Wallace, S. S., and Doublié, S. (2009) J. Biol. Chem. 284, 26174-26183). Here, we present two crystal structures of MvNei1 bound to two oxidized pyrimidines, Tg and 5-OHU. Both lesions are flipped out from the DNA helix. Tg is in the anti conformation, whereas 5-OHU adopts both anti and syn conformations in the glycosylase active site. Only two protein side chains (Glu-6 and Tyr-253) are within hydrogen-bonding contact with either damaged base, and mutating these residues did not markedly affect the glycosylase activity. This finding suggests that lesion recognition by Nei occurs before the damaged base flips into the glycosylase active site.
Asunto(s)
ADN Glicosilasas/química , ADN/química , Timina/análogos & derivados , Uracilo/análogos & derivados , Dominio Catalítico , Cristalografía por Rayos X , Humanos , Mimiviridae/enzimología , Oxidación-Reducción , Timina/química , Uracilo/químicaRESUMEN
5-Hydroxycytosine (5-OHC) is a stable oxidation product of cytosine associated with an increased frequency of C â T transition mutations. When this lesion escapes recognition by the base excision repair pathway and persists to serve as a templating base during DNA synthesis, replicative DNA polymerases often misincorporate dAMP at the primer terminus, which can lead to fixation of mutations and subsequent disease. To characterize the dynamics of DNA synthesis opposite 5-OHC, we initiated a comparison of unmodified dCMP to 5-OHC, 5-fluorocytosine (5-FC), and 5-methylcytosine (5-MEC) in which these bases act as templates in the active site of RB69 gp43, a high-fidelity DNA polymerase sharing homology with human replicative DNA polymerases. This study presents the first crystal structure of any DNA polymerase binding this physiologically important premutagenic DNA lesion, showing that while dGMP is stabilized by 5-OHC through normal Watson-Crick base pairing, incorporation of dAMP leads to unstacking and instability in the template. Furthermore, the electronegativity of the C5 substituent appears to be important in the miscoding potential of these cytosine-like templates. While dAMP is incorporated opposite 5-OHC ~5 times more efficiently than opposite unmodified dCMP, an elevated level of incorporation is also observed opposite 5-FC but not 5-MEC. Taken together, these data imply that the nonuniform templating by 5-OHC is due to weakened stacking capabilities, which allows dAMP incorporation to proceed in a manner similar to that observed opposite abasic sites.
Asunto(s)
Bacteriófagos/enzimología , Citosina/análogos & derivados , Replicación del ADN , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismo , Bacteriófagos/química , Emparejamiento Base , Dominio Catalítico , Citosina/química , Citosina/metabolismo , ADN Polimerasa Dirigida por ADN/genética , Moldes Genéticos , Proteínas Virales/genéticaRESUMEN
ATLAS (Any Target Ligand Affinity Screen) (Anadys Pharmaceuticals, Inc., San Diego, CA) is a homogeneous, affinity-based high-throughput screening technology based on protein thermal denaturation and the ability of ligands to bind and stabilize the target protein from unfolding. To further understand the assay sensitivity for the identification of ligands that bind to soluble protein targets, firefly luciferase was chosen to characterize the technology. Luciferase is a multidomain protein with a complex unfolding pathway. Binding of ATP results in a stabilizing conformational rearrangement of the domains. Using luciferase to characterize the ATLAS technology allowed us to evaluate the generality of the screening method for the identification of ligand binding to any target. Luciferase inhibitors identified from functional screens were used to assess the capability of ATLAS to rank order inhibitors. Comparison of the ATLAS 50% effective concentration with other biophysical and biochemical methods offered insight into optimizing ATLAS assay conditions to maximize sensitivity to compound binding and protein stabilization. The results show the importance of characterizing the thermal unfolding and aggregation behavior of the protein to allow the ATLAS screen to be optimally designed.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Proteínas/química , Adenosina Trifosfato/farmacología , Fenómenos Biofísicos , Biofisica , Calibración , Calorimetría , Dicroismo Circular , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Genes Reporteros/genética , Histidina , Calor , Indicadores y Reactivos , Cinética , Ligandos , Luz , Luciferasas/antagonistas & inhibidores , Luciferasas/genética , Oligopéptidos , Conformación Proteica , Desnaturalización Proteica , Dispersión de RadiaciónRESUMEN
5-Hydroxy-3(2H)-pyridazinone derivatives were investigated as inhibitors of genotype 1 HCV NS5B polymerase. The structure-activity relationship (SAR) associated with variation of the pyridazinone 2- and 6-substituents is discussed. The synthesis and metabolic stability of this new class of compounds are also described.