RESUMO
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.
Assuntos
DNA Helicases/metabolismo , DNA de Cadeia Simples/metabolismo , Desoxirribonuclease I/metabolismo , Conformação de Ácido Nucleico , Conformação Proteica , Engenharia de Proteínas/métodos , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo , Transativadores/metabolismo , Proteínas Virais/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Circoviridae/enzimologia , Sequência Conservada , Cristalografia por Raios X , DNA Helicases/química , DNA de Cadeia Simples/química , Desoxirribonuclease I/química , Biblioteca Gênica , Modelos Moleculares , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Vírus de Plantas/enzimologia , Ligação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Origem de Replicação , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Endonucleases Específicas para DNA e RNA de Cadeia Simples/química , Especificidade por Substrato , Transativadores/química , Proteínas Virais/químicaRESUMO
The Rep domain of Wheat dwarf virus (WDV Rep) is an HUH endonuclease involved in rolling-circle replication. HUH endonucleases coordinate a metal ion to enable the nicking of a specific ssDNA sequence and the subsequent formation of an intermediate phosphotyrosine bond. This covalent protein-ssDNA adduct makes HUH endonucleases attractive fusion tags (HUH-tags) in a diverse number of biotechnological applications. Solving the structure of an HUH endonuclease in complex with ssDNA will provide critical information about ssDNA recognition and sequence specificity, thus enabling rationally engineered protein-DNA interactions that are programmable. The structure of the WDV Rep domain reported here was solved in the apo state from a crystal diffracting to 1.24â Å resolution and represents an initial step in the direction of solving the structure of a protein-ssDNA complex.
