HUH-tag

HUH endonucleases are broadly split into two categories of enzymes: replication initiator proteins (Rep) or relaxase / mobilization proteins. They both contain small protein domains that recognize sequence-specific origins of replication or origin of transfer at which site they nick DNA. The nicking domain of Reps tend to be smaller, on the order of 10-20 kDa while nicking domains from relaxases are larger, roughly 20-40 kDa in size.[2]

HUH endonucleases generally have two histidine (H) residues in the active site coordinating a metal cation (Mg²⁺ or Mn²⁺) that interacts with the phosphate backbone of DNA. This allows for a nucleophilic attack, most commonly, by an activated tyrosine of the scissile phosphate in the DNA backbone, generating a 5' covalent bond with the ssDNA. In contrast to other DNA-protein linkage approaches, this reaction occurs at ambient conditions and does not require any additional modifications. X-ray crystallography and NMR structures have provided insight into the sequence specificity of DNA binding.[3][4]

WDV Rep reaction mechanism of nucleophilic attack on the DNA backbone.

• MobA relaxase incorporated into the viral capsid of Adeno-associated virus to link a DNA-antibody conjugate to target the virus to specific cell types[5]
• PCV2 Rep protein fused to Cas9 to covalently link a DNA repair template to Cas9, resulting in increased homology-directed repair in human cells[6]
• Similar to the approach mentioned above, Agrobacterium VirD2 relaxase fused to Cas9 allowing for linking of a DNA repair template to increase homology-directed repair in plants[7]
• PCV2 Rep protein fused to Elastin-like particles (ELPs) linked to a Mucin-1 DNA aptamer to deliver drugs to cancer cells[8]
• TraI, MobA, and TrwC relaxases used in orthogonal assembly on DNA nanostructures[9]
• PCV2 Rep protein fused to luciferase linked to DNA aptamers that detected thrombin levels in a sample[10]