RESUMEN
The base excision repair (BER) pathway is essential for the removal of DNA bases damaged by alkylation or oxidation. A key step in BER is the processing of an apurinic/apyrimidinic (AP) site intermediate by an AP endonuclease. The major AP endonuclease in human cells (APE1, also termed HAP1 and Ref-1) accounts for >95% of the total AP endonuclease activity, and is essential for the protection of cells against the toxic effects of several classes of DNA damaging agents. Moreover, APE1 overexpression has been linked to radio- and chemo-resistance in human tumors. Using a newly developed high-throughput screen, several chemical inhibitors of APE1 have been isolated. Amongst these, CRT0044876 was identified as a potent and selective APE1 inhibitor. CRT0044876 inhibits the AP endonuclease, 3'-phosphodiesterase and 3'-phosphatase activities of APE1 at low micromolar concentrations, and is a specific inhibitor of the exonuclease III family of enzymes to which APE1 belongs. At non-cytotoxic concentrations, CRT0044876 potentiates the cytotoxicity of several DNA base-targeting compounds. This enhancement of cytotoxicity is associated with an accumulation of unrepaired AP sites. In silico modeling studies suggest that CRT0044876 binds to the active site of APE1. These studies provide both a novel reagent for probing APE1 function in human cells, and a rational basis for the development of APE1-targeting drugs for antitumor therapy.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Reparación del ADN/efectos de los fármacos , ADN-(Sitio Apurínico o Apirimidínico) Liasa/antagonistas & inhibidores , Indoles/química , Indoles/farmacología , Línea Celular Tumoral , ADN-(Sitio Apurínico o Apirimidínico) Liasa/química , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Modelos MolecularesRESUMEN
Developing small-molecule antagonists against protein-protein interactions will provide powerful tools for mechanistic/functional studies and the discovery of new antibacterials. We have developed a reverse yeast three-hybrid approach that allows high-throughput screening for antagonist peptides against essential protein-protein interactions. We have applied our methodology to the essential bacterial helicase-primase interaction in Bacillus stearothermophilus and isolated a unique antagonist peptide. This peptide binds to the primase, thus excluding the helicase and inhibiting an essential interaction in bacterial DNA replication. We provide proof of principle that our reverse yeast three-hybrid method is a powerful "one-step" screen tool for direct high-throughput antagonist peptide selection against any protein-protein interaction detectable by traditional yeast two-hybrid systems. Such peptides will provide useful "leads" for the development of new antibacterials.
Asunto(s)
ADN Helicasas/metabolismo , ADN Primasa/metabolismo , Fragmentos de Péptidos/metabolismo , Péptidos/metabolismo , Técnicas del Sistema de Dos Híbridos , Adenosina Trifosfatasas/metabolismo , Proteínas Bacterianas/metabolismo , Unión Competitiva , Geobacillus stearothermophilus/enzimología , Fragmentos de Péptidos/síntesis química , Biblioteca de Péptidos , Péptidos/síntesis química , Unión Proteica , Saccharomyces cerevisiaeRESUMEN
Loading of the replicative ring helicase onto the origin of replication (oriC) is the final outcome of a well coordinated series of events that collectively constitute a primosomal cascade. Once the ring helicase is loaded, it recruits the primase and signals the switch to the polymerization mode. The transient nature of the helicase-primase (DnaB-DnaG) interaction in the Escherichia coli system has hindered our efforts to elucidate its structure and function. Taking advantage of the stable DnaB-DnaG complex in Bacillus stearothermophilus, we have reviewed conflicting mutagenic data from other bacterial systems and shown that DnaG interacts with the flexible linker that connects the N- and C-terminal domains of DnaB. Furthermore, atomic force microscopy (AFM) imaging experiments show that binding of the primase to the helicase induces predominantly a 3-fold symmetric morphology to the hexameric ring. Overall, three DnaG molecules appear to interact with the hexameric ring helicase but a small number of complexes with two and even one DnaG molecule bound to DnaB were also detected. The structural/functional significance of these data is discussed and a speculative structural model for this complex is suggested.
Asunto(s)
ADN Helicasas/química , ADN Helicasas/metabolismo , ADN Primasa/metabolismo , Geobacillus stearothermophilus/enzimología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , ADN Helicasas/genética , ADN Helicasas/ultraestructura , ADN Primasa/ultraestructura , Replicación del ADN , AdnB Helicasas , Geobacillus stearothermophilus/genética , Microscopía de Fuerza Atómica , Modelos Moleculares , Mutación/genética , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
The clamp-loader-helicase interaction is an important feature of the replisome. Although significant biochemical and structural work has been carried out on the clamp-loader-clamp-DNA polymerase alpha interactions in Escherichia coli, the clamp-loader-helicase interaction is poorly understood by comparison. The tau subunit of the clamp-loader mediates the interaction with DnaB. We have recently characterised this interaction in the Bacillus system and established a tau(5)-DnaB(6) stoichiometry. Here, we have obtained atomic force microscopy images of the tau-DnaB complex that reveal the first structural insight into its architecture. We show that despite the reported absence of the shorter gamma version in Bacillus, tau has a domain organisation similar to its E.coli counterpart and possesses an equivalent C-terminal domain that interacts with DnaB. The interaction interface of DnaB is also localised in its C-terminal domain. The combined data contribute towards our understanding of the bacterial replisome.