HUH-tag


HUH endonucleases are sequence-specific single-stranded DNA binding proteins originating from numerous species of bacteria and viruses. Viral HUH endonucleases are naturally involved in initiating rolling circle replication while ones of bacterial origin initiate bacterial conjugation. In biotechnology, they can be used to create protein-DNA linkages, akin to other methods such as SNAP-tag. In doing so, they create a 5' covalent bond between the ssDNA and the protein. HUH endonucleases can be fused with other proteins or used as protein tags.

Types of HUH endonucleases

HUH endonucleases are broadly split into two categories of enzymes: replication initiator proteins 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.

Mode of action

HUH endonucleases generally have two histidine residues in the active site coordinating a metal cation 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.

Applications