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

PloS One
Leticia AgúndezEls Henckaerts

Abstract

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 ...Continue Reading

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Citations

Jan 1, 2020·Cellular Microbiology·Sophie BlevesMatxalen Llosa
Aug 28, 2020·Frontiers in Molecular Biosciences·Itxaso Álvarez-RodríguezItziar Alkorta

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Methods Mentioned

BETA
PCR
gel filtration
transfection
flow citometry
helicase
Fluorescence

Software Mentioned

Image Quant TL
Openlab
Improvision
PyMOL Molecular Graphics System
Image Lab
SEDFIT

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