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 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,[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.

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 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.

Applications

References

  1. Chandler, Michael; de la Cruz, Fernando; Dyda, Fred; Hickman, Alison B.; Moncalian, Gabriel; Ton-Hoang, Bao (2013-07-08). "Breaking and joining single-stranded DNA: the HUH endonuclease superfamily". Nature Reviews Microbiology. 11 (8): 525–538. doi:10.1038/nrmicro3067. ISSN 1740-1526. PMC 6493337. PMID 23832240.
  2. Lovendahl, Klaus N.; Hayward, Amanda N.; Gordon, Wendy R. (2017-05-24). "Sequence-Directed Covalent Protein–DNA Linkages in a Single Step Using HUH-Tags". Journal of the American Chemical Society. 139 (20): 7030–7035. doi:10.1021/jacs.7b02572. ISSN 0002-7863. PMC 5517037. PMID 28481515.
  3. 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. ISSN 0022-2836. PMID 17275023.
  4. Everett, Blake A.; Litzau, Lauren A.; Tompkins, Kassidy; Shi, Ke; Nelson, Andrew; Aihara, Hideki; Evans Iii, Robert L.; Gordon, Wendy R. (2019-12-01). "Crystal structure of the Wheat dwarf virus Rep domain". Acta Crystallographica Section F. 75 (Pt 12): 744–749. doi:10.1107/S2053230X19015796. ISSN 2053-230X. PMC 6891580. PMID 31797816.
  5. Zdechlik, Alina C.; He, Yungui; Aird, Eric J.; Gordon, Wendy R.; Schmidt, Daniel (2019-12-06). "Programmable Assembly of Adeno-Associated Virus–Antibody Composites for Receptor-Mediated Gene Delivery". Bioconjugate Chemistry. 31 (4): 1093–1106. doi:10.1021/acs.bioconjchem.9b00790. ISSN 1043-1802. PMC 7676631. PMID 31809024.
  6. Aird, Eric J.; Lovendahl, Klaus N.; Martin, Amber St; Harris, Reuben S.; Gordon, Wendy R. (2018-05-31). "Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template". Communications Biology. 1 (1): 54. doi:10.1038/s42003-018-0054-2. ISSN 2399-3642. PMC 6123678. PMID 30271937.
  7. Ali, Zahir; Shami, Ashwag; Sedeek, Khalid; Kamel, Radwa; Alhabsi, Abdulrahman; Tehseen, Muhammad; Hassan, Norhan; Butt, Haroon; Kababji, Ahad; Hamdan, Samir M.; Mahfouz, Magdy M. (2020-01-23). "Fusion of the Cas9 endonuclease and the VirD2 relaxase facilitates homology-directed repair for precise genome engineering in rice". Communications Biology. 3 (1): 44. doi:10.1038/s42003-020-0768-9. ISSN 2399-3642. PMC 6978410. PMID 31974493.
  8. 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. ISSN 0957-4484. PMID 32176872.
  9. Sagredo, Sandra; Pirzer, Tobias; Aghebat Rafat, Ali; Goetzfried, Marisa A.; Moncalian, Gabriel; Simmel, Friedrich C.; de la Cruz, Fernando (2016). "Orthogonal Protein Assembly on DNA Nanostructures Using Relaxases". Angewandte Chemie International Edition. 55 (13): 4348–4352. doi:10.1002/anie.201510313. ISSN 1521-3773. PMC 5067690. PMID 26915475.
  10. Mie, Masayasu; Niimi, Takahiro; Mashimo, Yasumasa; Kobatake, Eiry (2019-01-03). "Construction of DNA-NanoLuc luciferase conjugates for DNA aptamer-based sandwich assay using Rep protein". Biotechnology Letters. 41 (3): 357–362. doi:10.1007/s10529-018-02641-7. ISSN 0141-5492. PMID 30603832.
This article is issued from Wikipedia. The text is licensed under Creative Commons - Attribution - Sharealike. Additional terms may apply for the media files.