HUH-tag
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.
The name HUH stands for "histidine-hydrophobic-histidine," referring to the three amino acids at the active site of the endonuclease. Some DNA viruses code for an HUH endonuclease which initiates rolling circle replication of the viral genome, and this process defines the realm Monodnaviria.[3]
Types of HUH endonucleases
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]
Mode of action
HUH endonucleases generally have two
Applications
- 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]
- PCV2 Rep protein fused to
- TraI, MobA, and TrwC relaxases used in orthogonal assembly on DNA nanostructures[10]
- PCV2 Rep protein fused to luciferase linked to DNA aptamers that detected thrombin levels in a sample[11]
References
- PMID 23832240.
- ^ PMID 28481515.
- ^ Koonin EV, Dolja VV, Krupovic M, Varsani A, Wolf YI, Yutin N, Zerbini M, Kuhn JH (18 October 2019). "Create a megataxonomic framework, filling all principal taxonomic ranks, for ssDNA viruses" (docx). International Committee on Taxonomy of Viruses. Retrieved 27 May 2020.
- PMID 17275023.
- PMID 31797816.
- PMID 31809024.
- PMID 30271937.
- PMID 31974493.
- PMID 32176872.
- PMID 26915475.
- PMID 30603832.