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.
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 1.24 Å resolution and represents an initial step in the direction of solving the structure of a protein-ssDNA complex.
Advanced biological molecule force probing methods such as atomic force microscopy and optical tweezers used to quantify forces at the single-molecule level are expensive and require extensive training and technical knowledge. However, the technologies underlying a centrifuge force microscope (CFM) are relatively straight forward, allowing for construction by labs with relatively low budgets and minimal training. Design ideas from previously constructed CFMs served as a guide in the development of this CFM. There were two primary goals: first, to develop an inexpensive, functional CFM using off-the-shelf and 3D printed parts; and second, to do so in the context of providing an educational experience for a broad range of students. The team included high school students and undergraduates from local high schools, the University of Minnesota, and other local higher education institutions. This project created an environment for student-focused development of the CFM that fostered active learning, individual ownership, as well as excellence in research. The instrument discussed herein represents a fully functional CFM designed and built by a postdoctoral researcher and a graduate student who together mentored several high school and undergraduate students.STATEMENT OF SIGNIFICANCEThe presented centrifuge force microscope (CFM) builds on features of existing designs specifically engineered for probing macromolecular force interactions at the single-molecule level. In the coming years, more versatile and modular CFM designs will be utilized in the force spectroscopy field, and the presented design is a step in that direction. In addition to constructing a functional instrument, true student ownership of the project design was equally an end goal. Students from high school through graduate school were included, and the project was structured so that everyone was seen as peers. This active learning project allowed students to acquire scientific concepts and techniques and apply them to real-life situations.
The wheat dwarf virus Rep domain is an HUH‐endonuclease and is involved in rolling‐circle replication. HUH‐endonucleases, or HUH‐tags, form covalent protein‐ssDNA adducts by coordinating a divalent metal ion to cleave a specific ssDNA sequence and form a phosphotyrosine linkage. This protein‐ssDNA fusion is useful for various biotechnology applications such as cellular imaging, cellular barcoding, DNA‐guided protein localization, and single molecule manipulation of DNA‐tethered proteins. Solving the structure of the Rep domain in complex with DNA could present necessary information regarding HUH‐tag sequence specificity and allow for rational engineering of protein‐DNA interactions. Here, the structure of WDV Rep domain in the apo state was solved with a crystal diffracting to 1.24 Å. While ssDNA soaks were attempted, they proved ineffective. However, the solved structure represents a step towards solving protein‐ssDNA complex. Support or Funding Information 1. National Institutes of Health/National Institute of General Medical Sciences (grant No. GM119483); NIH (grant No.242 NIGMS R35‐GM118047)
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