HUH

endonuclease Endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain. Some, such as deoxyribonuclease I, cut DNA relatively nonspecifically (without regard to sequence), while many, typically called restriction endonucleases ...

s (HUH-tags) are sequence-specific single-stranded DNA (ssDNA) binding proteins originating from numerous species of bacteria and viruses. Viral HUH endonucleases are involved in initiating

rolling circle replication Rolling circle replication (RCR) is a process of unidirectional nucleic acid replication that can rapidly synthesize multiple copies of circular molecules of DNA or RNA, such as plasmids, the genomes of bacteriophages, and the circular RNA genom ...

while ones of bacterial origin initiate

bacterial conjugation Bacterial conjugation is the transfer of genetic material between bacterial cells by direct cell-to-cell contact or by a bridge-like connection between two cells. This takes place through a pilus. It is a parasexual mode of reproduction in bacteri ...

. In biotechnology, they can be used to create protein-DNA linkages, akin to other methods such as

SNAP-tag SNAP-tag® is a self-labeling protein tag commercially available in various expression vectors. SNAP-tag is a 182 residues polypeptide (19.4 kDa) that can be fused to any protein of interest and further specifically and covalently tagged with a sui ...

. In doing so, they create a 5'

covalent bond A covalent bond is a chemical bond that involves the sharing of electrons to form electron pairs between atoms. These electron pairs are known as shared pairs or bonding pairs. The stable balance of attractive and repulsive forces between atoms ...

between the ssDNA and the protein. HUH endonucleases can be fused with other proteins or used as

protein tag Protein tags are peptide sequences genetically grafted onto a recombinant protein. Tags are attached to proteins for various purposes. They can be added to either end of the target protein, so they are either C-terminus or N-terminus specific or a ...

Types of HUH endonucleases

HUH endonucleases are broadly split into two categories of

enzyme Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products. A ...

s: replication initiator proteins (Rep) or

relaxase A relaxase is a single-strand DNA transesterase enzyme produced by some prokaryotes and viruses. Relaxases are responsible for site- and strand-specific nicks in unwound double-stranded DNA . Known relaxases belong to the rolling circle replicati ...

/ mobilization proteins. They both contain small protein domains that recognize sequence-specific

origins of replication The origin of replication (also called the replication origin) is a particular sequence in a genome at which replication is initiated. Propagation of the genetic material between generations requires timely and accurate duplication of DNA by semi ...

origin of transfer An origin of transfer (''oriT'') is a short sequence ranging from 40-500 base pairs in length that is necessary for the transfer of DNA from a gram-negative bacterial donor to recipient during bacterial conjugation. The transfer of DNA is a critica ...

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 Histidine (symbol His or H) is an essential amino acid that is used in the biosynthesis of proteins. It contains an α-amino group (which is in the protonated –NH3+ form under biological conditions), a carboxylic acid group (which is in the de ...

(H) residues in the

active site In biology and biochemistry, the active site is the region of an enzyme where substrate molecules bind and undergo a chemical reaction. The active site consists of amino acid residues that form temporary bonds with the substrate (binding site) a ...

coordinating a metal

cation An ion () is an atom or molecule with a net electrical charge. The charge of an electron is considered to be negative by convention and this charge is equal and opposite to the charge of a proton, which is considered to be positive by convent ...

( Mg²⁺ or Mn²⁺) that interacts with the phosphate backbone of DNA. This allows for a

nucleophilic attack In chemistry, a nucleophile is a chemical species that forms bonds by donating an electron pair. All molecules and ions with a free pair of electrons or at least one pi bond can act as nucleophiles. Because nucleophiles donate electrons, they are ...

, most commonly, by an activated

tyrosine -Tyrosine or tyrosine (symbol Tyr or Y) or 4-hydroxyphenylalanine is one of the 20 standard amino acids that are used by cells to synthesize proteins. It is a non-essential amino acid with a polar side group. The word "tyrosine" is from the Gr ...

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 X-ray crystallography is the experimental science determining the atomic and molecular structure of a crystal, in which the crystalline structure causes a beam of incident X-rays to diffract into many specific directions. By measuring the angles ...

and

NMR Nuclear magnetic resonance (NMR) is a physical phenomenon in which nuclei in a strong constant magnetic field are perturbed by a weak oscillating magnetic field (in the near field) and respond by producing an electromagnetic signal with ...

structures have provided insight into the sequence specificity of DNA binding. WDV Rep cleavage mechanism

Applications

* MobA relaxase incorporated into the

viral capsid A capsid is the protein shell of a virus, enclosing its genetic material. It consists of several oligomeric (repeating) structural subunits made of protein called protomers. The observable 3-dimensional morphological subunits, which may or ma ...

Adeno-associated virus Adeno-associated viruses (AAV) are small viruses that infect humans and some other primate species. They belong to the genus ''Dependoparvovirus'', which in turn belongs to the family ''Parvoviridae''. They are small (approximately 26 nm in di ...

to link a DNA-antibody conjugate to target the virus to specific cell types * PCV2 Rep protein fused to

Cas9 Cas9 (CRISPR associated protein 9, formerly called Cas5, Csn1, or Csx12) is a 160 kilodalton protein which plays a vital role in the immunological defense of certain bacteria against DNA viruses and plasmids, and is heavily utilized in genetic e ...

to covalently link a DNA repair template to Cas9, resulting in increased

homology-directed repair Homology-directed repair (HDR) is a mechanism in cells to repair double-strand DNA lesions. The most common form of HDR is homologous recombination. The HDR mechanism can only be used by the cell when there is a homologous piece of DNA present ...

in human cells * Similar to the approach mentioned above,

Agrobacterium ''Agrobacterium'' is a genus of Gram-negative bacteria established by H. J. Conn that uses horizontal gene transfer to cause tumors in plants. ''Agrobacterium tumefaciens'' is the most commonly studied species in this genus. ''Agrobacterium'' is ...

VirD2

fused to Cas9 allowing for linking of a DNA repair template to increase homology-directed repair in plants *PCV2 Rep protein fused to Elastin-like particles (ELPs) linked to a

Mucin-1 Mucin-1 (MUC-1) is a heterodimer transmembrane protein of the mucin family encoded in humans by the ''MUC1'' gene. It is cleaved into two chains: Mucin-1 subunit alpha (MUC1-NT; MUC1-alpha) and Mucin-1 subunit beta (MUC-CT; MUC1-beta). These su ...

DNA

aptamer Aptamers are short sequences of artificial DNA, RNA, XNA, or peptide that bind a specific target molecule, or family of target molecules. They exhibit a range of affinities ( KD in the pM to μM range), with little or no off-target binding ...

to deliver drugs to cancer cells *TraI, MobA, and TrwC relaxases used in orthogonal assembly on DNA nanostructures *PCV2 Rep protein fused to

luciferase Luciferase is a generic term for the class of oxidative enzymes that produce bioluminescence, and is usually distinguished from a photoprotein. The name was first used by Raphaël Dubois who invented the words ''luciferin'' and ''luciferase'', ...

linked to DNA aptamers that detected

thrombin Thrombin (, ''fibrinogenase'', ''thrombase'', ''thrombofort'', ''topical'', ''thrombin-C'', ''tropostasin'', ''activated blood-coagulation factor II'', ''blood-coagulation factor IIa'', ''factor IIa'', ''E thrombin'', ''beta-thrombin'', ''gamma- ...

levels in a sample{{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

References

DNA-binding proteins