View source for HUH-tag

[[File:HUH reaction.tif|thumb|300x300px|Generic HUH endonuclease binding to single-stranded DNA.]]
'''HUH [[endonuclease]]s''' (HUH-tags) are sequence-specific single-stranded [[DNA]] (ssDNA) [[DNA-binding protein|binding proteins]] originating from numerous species of bacteria and viruses.<ref>{{Cite journal|last=Chandler|first=Michael|last2=de la Cruz|first2=Fernando|last3=Dyda|first3=Fred|last4=Hickman|first4=Alison B.|last5=Moncalian|first5=Gabriel|last6=Ton-Hoang|first6=Bao|date=2013-07-08|title=Breaking and joining single-stranded DNA: the HUH endonuclease superfamily|journal=Nature Reviews Microbiology|volume=11|issue=8|pages=525–538|doi=10.1038/nrmicro3067|pmid=23832240|pmc=6493337|issn=1740-1526}}</ref> 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,<ref name=":0">{{Cite journal|last=Lovendahl|first=Klaus N.|last2=Hayward|first2=Amanda N.|last3=Gordon|first3=Wendy R.|date=2017-05-24|title=Sequence-Directed Covalent Protein–DNA Linkages in a Single Step Using HUH-Tags|journal=Journal of the American Chemical Society|volume=139|issue=20|pages=7030–7035|doi=10.1021/jacs.7b02572|issn=0002-7863|pmc=5517037|pmid=28481515}}</ref> 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 [[Fusion protein|fused]] with other proteins or used as [[protein tag]]s.
The name HUH stands for "[[histidine]]-hydrophobic-histidine," referring to the three amino acids at the [[active site]] of the endonuclease. Some [[DNA virus]]es code for an HUH endonuclease which initiates [[rolling circle replication]] of the viral genome, and this process defines the [[realm (virology)|realm]] ''[[Monodnaviria]]''.<ref>{{cite web|vauthors=Koonin EV, Dolja VV, Krupovic M, Varsani A, Wolf YI, Yutin N, Zerbini M, Kuhn JH|title=Create a megataxonomic framework, filling all principal taxonomic ranks, for ssDNA viruses|url=https://ictv.global/ictv/proposals/2019.005G.zip|website=International Committee on Taxonomy of Viruses|access-date=27 May 2020|language=en|format=docx|date=18 October 2019}}</ref>
== Types of HUH endonucleases ==
HUH endonucleases are broadly split into two categories of [[enzyme]]s: replication initiator proteins (Rep) or [[relaxase]] / mobilization proteins. They both contain small protein domains that recognize sequence-specific [[Origin of replication|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.<ref name=":0" />
== Mode of action ==
HUH endonucleases generally have two [[histidine]] (H) residues in the [[active site]] coordinating a metal [[cation]] ([[Mg²⁺|Mg<sup>2+</sup>]] or [[Mn²⁺|Mn<sup>2+</sup>]]) 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 [[Nuclear magnetic resonance|NMR]] structures have provided insight into the sequence specificity of DNA binding.<ref>{{Cite journal|last=Vega-Rocha|first=Susana|last2=Byeon|first2=In-Ja L.|last3=Gronenborn|first3=Bruno|last4=Gronenborn|first4=Angela M.|last5=Campos-Olivas|first5=Ramón|date=2007|title=Solution Structure, Divalent Metal and DNA Binding of the Endonuclease Domain from the Replication Initiation Protein from Porcine Circovirus 2|journal=Journal of Molecular Biology|volume=367|issue=2|pages=473–487|doi=10.1016/j.jmb.2007.01.002|pmid=17275023|issn=0022-2836}}</ref><ref>{{Cite journal|last=Everett|first=Blake A.|last2=Litzau|first2=Lauren A.|last3=Tompkins|first3=Kassidy|last4=Shi|first4=Ke|last5=Nelson|first5=Andrew|last6=Aihara|first6=Hideki|last7=Evans Iii|first7=Robert L.|last8=Gordon|first8=Wendy R.|date=2019-12-01|title=Crystal structure of the Wheat dwarf virus Rep domain|journal=Acta Crystallographica Section F|volume=75|issue=Pt 12|pages=744–749|doi=10.1107/S2053230X19015796|issn=2053-230X|pmc=6891580|pmid=31797816}}</ref>
[[File:WDV Rep cleavage mechanism.png|alt=|center|thumb|880x880px|WDV Rep reaction mechanism of nucleophilic attack on the DNA backbone.]]
== Applications ==
* MobA relaxase incorporated into the [[Capsid|viral capsid]] of [[Adeno-associated virus]] to link a DNA-antibody conjugate to target the virus to specific cell types<ref>{{Cite journal|last=Zdechlik|first=Alina C.|last2=He|first2=Yungui|last3=Aird|first3=Eric J.|last4=Gordon|first4=Wendy R.|last5=Schmidt|first5=Daniel|date=2019-12-06|title=Programmable Assembly of Adeno-Associated Virus–Antibody Composites for Receptor-Mediated Gene Delivery|journal=Bioconjugate Chemistry|volume=31|issue=4|pages=1093–1106|doi=10.1021/acs.bioconjchem.9b00790|pmid=31809024|issn=1043-1802|pmc=7676631}}</ref>
*[[Porcine circovirus|PCV2]] Rep protein fused to [[Cas9]] to covalently link a DNA repair template to Cas9, resulting in increased [[Homology directed repair|homology-directed repair]] in human cells<ref>{{Cite journal|last=Aird|first=Eric J.|last2=Lovendahl|first2=Klaus N.|last3=Martin|first3=Amber St|last4=Harris|first4=Reuben S.|last5=Gordon|first5=Wendy R.|date=2018-05-31|title=Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template|journal=Communications Biology|language=en|volume=1|issue=1|pages=54|doi=10.1038/s42003-018-0054-2|issn=2399-3642|pmc=6123678|pmid=30271937}}</ref>
* 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<ref>{{Cite journal|last=Ali|first=Zahir|last2=Shami|first2=Ashwag|last3=Sedeek|first3=Khalid|last4=Kamel|first4=Radwa|last5=Alhabsi|first5=Abdulrahman|last6=Tehseen|first6=Muhammad|last7=Hassan|first7=Norhan|last8=Butt|first8=Haroon|last9=Kababji|first9=Ahad|last10=Hamdan|first10=Samir M.|last11=Mahfouz|first11=Magdy M.|date=2020-01-23|title=Fusion of the Cas9 endonuclease and the VirD2 relaxase facilitates homology-directed repair for precise genome engineering in rice|journal=Communications Biology|language=en|volume=3|issue=1|pages=44|doi=10.1038/s42003-020-0768-9|issn=2399-3642|pmc=6978410|pmid=31974493}}</ref>
*PCV2 Rep protein fused to [[Elastin-like polypeptides|Elastin-like particles]] (ELPs) linked to a [[MUC1|Mucin-1]] DNA [[aptamer]] to deliver drugs to cancer cells<ref>{{Cite journal|last=Guo|first=Wei|last2=Mashimo|first2=Yasumasa|last3=Kobatake|first3=Eiry|last4=Mie|first4=Masayasu|date=2020-03-16|title=Construction of DNA-displaying nanoparticles by enzymatic conjugation of DNA and elastin-like polypeptides using a replication initiation protein|journal=Nanotechnology|volume=31|issue=25|pages=255102|doi=10.1088/1361-6528/ab8042|pmid=32176872|issn=0957-4484}}</ref>
*TraI, MobA, and TrwC relaxases used in orthogonal assembly on [[DNA origami|DNA nanostructures]]<ref>{{Cite journal|last=Sagredo|first=Sandra|last2=Pirzer|first2=Tobias|last3=Aghebat Rafat|first3=Ali|last4=Goetzfried|first4=Marisa A.|last5=Moncalian|first5=Gabriel|last6=Simmel|first6=Friedrich C.|last7=de la Cruz|first7=Fernando|date=2016|title=Orthogonal Protein Assembly on DNA Nanostructures Using Relaxases|journal=Angewandte Chemie International Edition|language=en|volume=55|issue=13|pages=4348–4352|doi=10.1002/anie.201510313|issn=1521-3773|pmc=5067690|pmid=26915475}}</ref>
*PCV2 Rep protein fused to [[luciferase]] linked to DNA aptamers that detected [[thrombin]] levels in a sample<ref>{{Cite journal|last=Mie|first=Masayasu|last2=Niimi|first2=Takahiro|last3=Mashimo|first3=Yasumasa|last4=Kobatake|first4=Eiry|date=2019-01-03|title=Construction of DNA-NanoLuc luciferase conjugates for DNA aptamer-based sandwich assay using Rep protein|journal=Biotechnology Letters|volume=41|issue=3|pages=357–362|doi=10.1007/s10529-018-02641-7|pmid=30603832|issn=0141-5492}}</ref>
*"Click editors" - PCV2 Rep protein fused to nCas9 and a DNA polymerase fragment to allow for templated genome editing<ref>{{Cite journal |last=Ferreira da Silva |first=Joana |last2=Tou |first2=Connor J. |last3=King |first3=Emily M. |last4=Eller |first4=Madeline L. |last5=Rufino-Ramos |first5=David |last6=Ma |first6=Linyuan |last7=Cromwell |first7=Christopher R. |last8=Metovic |first8=Jasna |last9=Benning |first9=Friederike M. C. |last10=Chao |first10=Luke H. |last11=Eichler |first11=Florian S. |last12=Kleinstiver |first12=Benjamin P. |date=2024-07-22 |title=Click editing enables programmable genome writing using DNA polymerases and HUH endonucleases |url=https://www.nature.com/articles/s41587-024-02324-x |journal=Nature Biotechnology |language=en |pages=1–13 |doi=10.1038/s41587-024-02324-x |issn=1546-1696}}</ref>
== References ==
<references />
[[Category:DNA-binding proteins]]