ABSTRACT
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.
Subject(s)
DNA Helicases/metabolism , DNA, Single-Stranded/metabolism , Deoxyribonuclease I/metabolism , Nucleic Acid Conformation , Protein Conformation , Protein Engineering/methods , Single-Strand Specific DNA and RNA Endonucleases/metabolism , Trans-Activators/metabolism , Viral Proteins/metabolism , Amino Acid Motifs , Amino Acid Sequence , Circoviridae/enzymology , Conserved Sequence , Crystallography, X-Ray , DNA Helicases/chemistry , DNA, Single-Stranded/chemistry , Deoxyribonuclease I/chemistry , Gene Library , Models, Molecular , Molecular Docking Simulation , Molecular Sequence Data , Plant Viruses/enzymology , Protein Binding , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Replication Origin , Sequence Alignment , Sequence Homology, Amino Acid , Single-Strand Specific DNA and RNA Endonucleases/chemistry , Substrate Specificity , Trans-Activators/chemistry , Viral Proteins/chemistryABSTRACT
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.
