RESUMEN
Monoclonal antibodies (mAbs) can be potent and highly specific therapeutics, diagnostics and research reagents. Nonetheless, mAb discovery using current in vivo or in vitro approaches can be costly and time-consuming, with no guarantee of success. We have established a platform for rapid discovery and optimization of mAbs ex vivo. This DTLacO platform derives from a chicken B cell line that has been engineered to enable rapid selection and seamless maturation of high affinity mAbs. We have validated the DTLacO platform by generation of high affinity and specific mAbs to five cell surface targets, the receptor tyrosine kinases VEGFR2 and TIE2, the glycoprotein TROP2, the small TNF receptor family member FN14, and the G protein-coupled receptor FZD10. mAb discovery is rapid and humanization is straightforward, establishing the utility of the DTLacO platform for identification of mAbs for therapeutic and other applications.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Redes Reguladoras de Genes/genética , Operón Lac/genética , Represoras Lac/genética , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales Humanizados/química , Anticuerpos Monoclonales Humanizados/inmunología , Afinidad de Anticuerpos/inmunología , Línea Celular , Pollos , Células Clonales , Regiones Determinantes de Complementariedad/genética , Secuencia Conservada/genética , Ingeniería Genética , Humanos , Cadenas Pesadas de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/química , Región Variable de Inmunoglobulina/inmunología , Datos de Secuencia Molecular , Mutación/genética , Receptores de Superficie Celular/inmunología , Estreptavidina/inmunologíaRESUMEN
DNA glycosylases/AP lyases initiate repair of oxidized bases in the genomes of all organisms by excising these lesions and then cleaving the DNA strand at the resulting abasic (AP) sites and generate 3' phospho alpha,beta-unsaturated aldehyde (3' PUA) or 3' phosphate (3' P) terminus. In Escherichia coli, the AP-endonucleases (APEs) hydrolyze both 3' blocking groups (3' PUA and 3' P) to generate the 3'-OH termini needed for repair synthesis. In mammalian cells, the previously characterized DNA glycosylases, NTH1 and OGG1, produce 3' PUA, which is removed by the only AP-endonuclease, APE1. However, APE1 is barely active in removing 3' phosphate generated by the recently discovered mammalian DNA glycosylases NEIL1 and NEIL2. We showed earlier that the 3' phosphate generated by NEIL1 is efficiently removed by polynucleotide kinase (PNK) and not APE1. Here we show that the NEIL2-initiated repair of 5-hydroxyuracil (5-OHU) similarly requires PNK. We have also observed stable interaction between NEIL2 and other BER proteins DNA polymerase beta (Pol beta), DNA ligase IIIalpha (Lig IIIalpha) and XRCC1. In spite of their limited sequence homology, NEIL1 and NEIL2 interact with the same domains of Pol beta and Lig IIIalpha. Surprisingly, while the catalytically dispensable C-terminal region of NEIL1 is the common interacting domain, the essential N-terminal segment of NEIL2 is involved in analogous interaction. The BER proteins including NEIL2, PNK, Pol beta, Lig IIIalpha and XRCC1 (but not APE1) could be isolated as a complex from human cells, competent for repair of 5-OHU in plasmid DNA.
Asunto(s)
ADN Glicosilasas/metabolismo , Reparación del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , ADN/metabolismo , Polinucleótido 5'-Hidroxil-Quinasa/metabolismo , Animales , Línea Celular , Línea Celular Tumoral , ADN Glicosilasas/aislamiento & purificación , ADN Ligasa (ATP) , ADN Ligasas/aislamiento & purificación , ADN Ligasas/metabolismo , ADN Polimerasa beta/aislamiento & purificación , ADN Polimerasa beta/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/aislamiento & purificación , Humanos , Complejos Multiproteicos , Plásmidos/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa , Polinucleótido 5'-Hidroxil-Quinasa/aislamiento & purificación , Estructura Terciaria de Proteína , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Transfección , Técnicas del Sistema de Dos Híbridos , Proteínas de XenopusRESUMEN
Tyrosyl-DNA phosphodiesterase (Tdp1) catalyzes the hydrolysis of the tyrosyl-3' phosphate linkage found in topoisomerase I-DNA covalent complexes. The inherited disorder, spinocerebellar ataxia with axonal neuropathy (SCAN1), is caused by a H493R mutation in Tdp1. Contrary to earlier proposals that this disease results from a loss-of-function mutation, we show here that this mutation reduces enzyme activity approximately 25-fold and importantly causes the accumulation of the Tdp1-DNA covalent reaction intermediate. Thus, the attempted repair of topoisomerase I-DNA complexes by Tdp1 unexpectedly generates a new protein-DNA complex with an apparent half-life of approximately 13 min that, in addition to the unrepaired topoisomerase I-DNA complex, may interfere with transcription and replication in human cells and contribute to the SCAN1 phenotype. The analysis of Tdp1 mutant cell lines derived from SCAN1 patients reveals that they are hypersensitive to the topoisomerase I-specific anticancer drug camptothecin (CPT), implicating Tdp1 in the repair of CPT-induced topoisomerase I damage in human cells. This finding suggests that inhibitors of Tdp1 could act synergistically with CPT in anticancer therapy.
Asunto(s)
Camptotecina/farmacología , Inhibidores Enzimáticos/farmacología , Hidrolasas Diéster Fosfóricas/genética , Sustitución de Aminoácidos , Citometría de Flujo , Humanos , Mutación , Neoplasias/tratamiento farmacológico , Hidrolasas Diéster Fosfóricas/metabolismo , Fase S/efectos de los fármacosRESUMEN
Human DNA topoisomerase I is an essential enzyme involved in resolving the torsional stress associated with DNA replication, transcription, and chromatin condensation. The catalytic cycle of the enzyme consists of DNA cleavage to form a covalent enzyme-DNA intermediate, DNA relaxation, and finally, re-ligation of the phosphate backbone to restore the continuity of the DNA. Structure/function studies have elucidated a flexible enzyme that relaxes DNA through coordinated, controlled movements of distinct enzyme domains. The cellular roles of topoisomerase I are apparent throughout the nucleus, but the concentration of processes acting on ribosomal DNA results in topoisomerase I accumulation in the nucleolus. Although the activity of topoisomerase I is required in these processes, the enzyme can also have a deleterious effect on cells. In the event that the final re-ligation step of the reaction cycle is prevented, the covalent topoisomerase I-DNA intermediate becomes a toxic DNA lesion that must be repaired. The complexities of the relaxation reaction, the cellular roles, and the pathways that must exist to repair topoisomerase I-mediated DNA damage highlight the importance of continued study of this essential enzyme.
Asunto(s)
Daño del ADN , ADN-Topoisomerasas de Tipo I/metabolismo , Replicación del ADN , ADN-Topoisomerasas de Tipo I/química , ADN-Topoisomerasas de Tipo I/genética , Humanos , Modelos Biológicos , Modelos Moleculares , Conformación ProteicaRESUMEN
The paradigm for repair of oxidized base lesions in genomes via the base excision repair (BER) pathway is based on studies in Escherichia coli, in which AP endonuclease (APE) removes all 3' blocking groups (including 3' phosphate) generated by DNA glycosylase/AP lyases after base excision. The recently discovered mammalian DNA glycosylase/AP lyases, NEIL1 and NEIL2, unlike the previously characterized OGG1 and NTH1, generate DNA strand breaks with 3' phosphate termini. Here we show that in mammalian cells, removal of the 3' phosphate is dependent on polynucleotide kinase (PNK), and not APE. NEIL1 stably interacts with other BER proteins, DNA polymerase beta (pol beta) and DNA ligase IIIalpha. The complex of NEIL1, pol beta, and DNA ligase IIIalpha together with PNK suggests coordination of NEIL1-initiated repair. That NEIL1/PNK could also repair the products of other DNA glycosylases suggests a broad role for this APE-independent BER pathway in mammals.
Asunto(s)
ADN Glicosilasas/metabolismo , Reparación del ADN/fisiología , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , ADN/metabolismo , Células Cultivadas , ADN Ligasa (ATP) , ADN Ligasas/metabolismo , ADN Polimerasa beta/metabolismo , Humanos , Proteínas de Unión a Poli-ADP-Ribosa , Polinucleótido 5'-Hidroxil-Quinasa/metabolismo , Saccharomyces cerevisiae , Técnicas del Sistema de Dos Híbridos , Proteínas de XenopusRESUMEN
The repair of DNA single-strand breaks in mammalian cells is mediated by poly(ADP-ribose) polymerase 1 (PARP-1), DNA ligase IIIalpha, and XRCC1. Since these proteins are not found in lower eukaryotes, this DNA repair pathway plays a unique role in maintaining genome stability in more complex organisms. XRCC1 not only forms a stable complex with DNA ligase IIIalpha but also interacts with several other DNA repair factors. Here we have used affinity chromatography to identify proteins that associate with DNA ligase III. PARP-1 binds directly to an N-terminal region of DNA ligase III immediately adjacent to its zinc finger. In further studies, we have shown that DNA ligase III also binds directly to poly(ADP-ribose) and preferentially associates with poly(ADP-ribosyl)ated PARP-1 in vitro and in vivo. Our biochemical studies have revealed that the zinc finger of DNA ligase III increases DNA joining in the presence of either poly(ADP-ribosyl)ated PARP-1 or poly(ADP-ribose). This provides a mechanism for the recruitment of the DNA ligase IIIalpha-XRCC1 complex to in vivo DNA single-strand breaks and suggests that the zinc finger of DNA ligase III enables this complex and associated repair factors to locate the strand break in the presence of the negatively charged poly(ADP-ribose) polymer.