Alanine scan of WDV Rep reveals its ssDNA binding mechanism

Abstract

HUH endonucleases (dubbed “HUH‐tags”) are small protein domains capable of forming covalent adducts with ssDNA in a sequence‐specific manner. The active site of HUH endonucleases utilizes a conserved histidine‐hydrophobic‐histidine motif to coordinate a metal ion that stabilizes a nucleophilic tyrosine necessary for phosphodiesterase activity. Because viral‐derived 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, wheat dwarf virus replication initiator protein (WDV), 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 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.