Interrogating mechanisms of ssDNA binding to a viral HUH-endonuclease by alanine scanning of an electrostatic patch

HUH endonucleases (dubbed "HUH-tags") are small protein domains capable of forming covalent adducts with ssDNA in a sequence-specific manner. Because viral HUH-tags are relatively small, react quickly, and require no chemical modifications to their ssDNA substrate, they have great value as protein fusion tags in biotechnologies ranging from genetic engineering to single-molecule studies. One of the greatest assets of these tags is sequence-specificity to their unique, native Ori sequence in vivo, introducing the possibility of using multiple HUH-tags in multiplexed "one-pot" reactions. However, their mechanism of ssDNA sequence binding and specificity is poorly understood, and there is noted cross-reactivity between tags of closely related species. In order to understand the mechanism of ssDNA binding, we performed an alanine scan along a positively-charged patch of one such HUH-tag, replication-associated protein from Wheat Dwarf Virus (WDV Rep), and characterized the enzymatic activity in both the rate and extent of the reaction. In molecular beacon Stopped-Flow experiments, single point mutants of WDV showed a more than 60% decrease in reaction rate constant, and gel shift assays showed an almost complete lack of activity for some variants for single nucleotide ssDNA substitutions. In all, these findings help allow us to highlight key interactions in WDV-ssDNA binding, and we gain further insight into potential rational engineering of HUH-endonucleases to bind desired sequences of DNA.