HUH endonucleases (HUH-tags) are sequence-specific single-stranded DNA (ssDNA) binding proteins originating from numerous species of bacteria and viruses.[1] Viral HUH endonucleases are 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,[2] 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.
Generic HUH endonuclease binding to single-stranded DNA.
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. These residues allow 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.[4][5]
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[6]
PCV2 Rep protein fused to Cas9 to covalently link a DNA repair template to Cas9, resulting in increased homology-directed repair in human cells[7]
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[8]
Vega-Rocha, Susana; Byeon, In-Ja L.; Gronenborn, Bruno; Gronenborn, Angela M.; Campos-Olivas, Ramón (2007). "Solution Structure, Divalent Metal and DNA Binding of the Endonuclease Domain from the Replication Initiation Protein from Porcine Circovirus 2". Journal of Molecular Biology. 367 (2): 473–487. doi:10.1016/j.jmb.2007.01.002. ISSN0022-2836. PMID17275023.
Guo, Wei; Mashimo, Yasumasa; Kobatake, Eiry; Mie, Masayasu (2020-03-16). "Construction of DNA-displaying nanoparticles by enzymatic conjugation of DNA and elastin-like polypeptides using a replication initiation protein". Nanotechnology. 31 (25): 255102. doi:10.1088/1361-6528/ab8042. ISSN0957-4484. PMID32176872.