Determination of Adeno-associated Virus Rep DNA Binding Using Fluorescence Anisotropy

Zárate‐Pérez, Francisco; Santosh, Vishaka; Bardelli, Martino; Agúndez, Leticia; Henckaerts, Els; Escalante, Carlos

doi:10.21769/bioprotoc.2194

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“…Samples were incubated at 25°C for 30 min prior to measurement. Binding activity was analyzed at an excitation of 492 nm and an emission of 520 nm [ 61 ]. Data analysis was performed as described by Yoon-Robarts et al .…”

Section: Methodsmentioning

confidence: 99%

Exchange of functional domains between a bacterial conjugative relaxase and the integrase of the human adeno-associated virus

et al. 2018

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Endonucleases of the HUH family are specialized in processing single-stranded DNA in a variety of evolutionarily highly conserved biological processes related to mobile genetic elements. They share a structurally defined catalytic domain for site-specific nicking and strand-transfer reactions, which is often linked to the activities of additional functional domains, contributing to their overall versatility. To assess if these HUH domains could be interchanged, we created a chimeric protein from two distantly related HUH endonucleases, containing the N-terminal HUH domain of the bacterial conjugative relaxase TrwC and the C-terminal DNA helicase domain of the human adeno-associated virus (AAV) replicase and site-specific integrase. The purified chimeric protein retained oligomerization properties and DNA helicase activities similar to Rep68, while its DNA binding specificity and cleaving-joining activity at oriT was similar to TrwC. Interestingly, the chimeric protein could catalyse site-specific integration in bacteria with an efficiency comparable to that of TrwC, while the HUH domain of TrwC alone was unable to catalyze this reaction, implying that the Rep68 C-terminal helicase domain is complementing the TrwC HUH domain to achieve site-specific integration into TrwC targets in bacteria. Our results illustrate how HUH domains could have acquired through evolution other domains in order to attain new roles, contributing to the functional flexibility observed in this protein superfamily.

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Section: Methodsmentioning

confidence: 99%