RESUMEN
Replication initiator proteins (Reps) from the HUH-endonuclease superfamily process specific single-stranded DNA (ssDNA) sequences to initiate rolling circle/hairpin replication in viruses, such as crop ravaging geminiviruses and human disease causing parvoviruses. In biotechnology contexts, Reps are the basis for HUH-tag bioconjugation and a critical adeno-associated virus genome integration tool. We solved the first co-crystal structures of Reps complexed to ssDNA, revealing a key motif for conferring sequence specificity and for anchoring a bent DNA architecture. In combination, we developed a deep sequencing cleavage assay, termed HUH-seq, to interrogate subtleties in Rep specificity and demonstrate how differences can be exploited for multiplexed HUH-tagging. Together, our insights allowed engineering of only four amino acids in a Rep chimera to predictably alter sequence specificity. These results have important implications for modulating viral infections, developing Rep-based genomic integration tools, and enabling massively parallel HUH-tag barcoding and bioconjugation applications.
Asunto(s)
ADN Helicasas/metabolismo , ADN de Cadena Simple/metabolismo , Desoxirribonucleasa I/metabolismo , Conformación de Ácido Nucleico , Conformación Proteica , Ingeniería de Proteínas/métodos , Endonucleasas Específicas del ADN y ARN con un Solo Filamento/metabolismo , Transactivadores/metabolismo , Proteínas Virales/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Circoviridae/enzimología , Secuencia Conservada , Cristalografía por Rayos X , ADN Helicasas/química , ADN de Cadena Simple/química , Desoxirribonucleasa I/química , Biblioteca de Genes , Modelos Moleculares , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Virus de Plantas/enzimología , Unión Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Origen de Réplica , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Endonucleasas Específicas del ADN y ARN con un Solo Filamento/química , Especificidad por Sustrato , Transactivadores/química , Proteínas Virales/químicaRESUMEN
The gut microbiome has been implicated in multiple human chronic gastrointestinal (GI) disorders. Determining its mechanistic role in disease has been difficult due to apparent disconnects between animal and human studies and lack of an integrated multi-omics view of disease-specific physiological changes. We integrated longitudinal multi-omics data from the gut microbiome, metabolome, host epigenome, and transcriptome in the context of irritable bowel syndrome (IBS) host physiology. We identified IBS subtype-specific and symptom-related variation in microbial composition and function. A subset of identified changes in microbial metabolites correspond to host physiological mechanisms that are relevant to IBS. By integrating multiple data layers, we identified purine metabolism as a novel host-microbial metabolic pathway in IBS with translational potential. Our study highlights the importance of longitudinal sampling and integrating complementary multi-omics data to identify functional mechanisms that can serve as therapeutic targets in a comprehensive treatment strategy for chronic GI diseases. VIDEO ABSTRACT.