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 major open reading frame of banana bunchy top virus (BBTV) DNA-1 encodes a putative replication initiation protein (Rep). In vitro, a fusion protein of BBTV Rep linked to a maltose-binding protein exhibited both site-specific nicking and joining activities. These activities were dependent on the presence of Mg2+ or Mn2+, but did not require ATP. The fusion protein specifically cleaved ssDNA between bases +7 and +8 of a conserved nonanucleotide loop sequence which is present in the virion-strand of the stem-loop common region of each BBTV component. During this reaction, the fusion protein became covalently attached to the 5' end of the 3'cleavage product. After the nicking reactions, the fusion protein was also capable of catalysing the joining of two nicked ssDNA fragments in a site-specific manner. Based on these activities, BBTV Rep would appear to be very similar to the Rep proteins of the geminiviruses.