HUH

endonuclease In molecular biology, endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain (namely DNA or RNA). Some, such as deoxyribonuclease I, cut DNA relatively nonspecifically (with regard to sequence), while man ...

s (HUH-tags) are sequence-specific single-stranded

DNA Deoxyribonucleic acid (; DNA) is a polymer composed of two polynucleotide chains that coil around each other to form a double helix. The polymer carries genetic instructions for the development, functioning, growth and reproduction of al ...

(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 Bacteria, 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 cycle, parasexual mode ...

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

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

-hydrophobic-histidine," referring to the three amino acids at 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, the ''binding s ...

of the endonuclease. Some

DNA virus A DNA virus is a virus that has a genome made of deoxyribonucleic acid (DNA) that is replicated by a DNA polymerase. They can be divided between those that have two strands of DNA in their genome, called double-stranded DNA (dsDNA) viruses, and t ...

es code for an HUH endonuclease which initiates

of the viral genome, and this process defines the

realm A realm is a community or territory over which a sovereign rules. The term is commonly used to describe a monarchical or dynastic state. A realm may also be a subdivision within an empire, if it has its own monarch, e.g. the German Empire. Etymo ...

Monodnaviria ''Monodnaviria'' is a Realm (virology), realm of viruses that includes all DNA virus#Group II: ssDNA viruses, single-stranded DNA viruses that Genetic code, encode an HUH-tag, endonuclease of the HUH superfamily that initiates rolling circle repli ...

''.

Types of HUH endonucleases

HUH endonucleases are broadly split into two categories of

enzyme An enzyme () is a protein that acts as a biological catalyst by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates into different mol ...

s: 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.

Mode of action

HUH endonucleases generally have two

(H) residues in the

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. These residues allow 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 a ...

, 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 conditionally essential amino acid with a polar side group. The word "tyrosine" is ...

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 of determining the atomic and molecular structure of a crystal, in which the crystalline structure causes a beam of incident X-rays to Diffraction, diffract in specific directions. By measuring th ...

and

NMR Nuclear magnetic resonance (NMR) is a physical phenomenon in which atomic nucleus, nuclei in a strong constant magnetic field are disturbed by a weak oscillating magnetic field (in the near and far field, near field) and respond by producing ...

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

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 * PCV2 Rep protein fused to

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

to covalently link a DNA repair template to Cas9, resulting in increased homology-directed repair in human cells * Similar to the approach mentioned above,

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

VirD2 relaxase 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 DNA

aptamer Aptamers are oligomers of artificial ssDNA, RNA, Xeno nucleic acid, XNA, or peptide that ligand, bind a specific target molecule, or family of target molecules. They exhibit a range of affinities (Dissociation constant, KD in the pM to μM rang ...

to deliver drugs to cancer cells *TraI, MobA, and TrwC relaxases used in orthogonal assembly on

DNA nanostructures Deoxyribonucleic acid (; DNA) is a polymer composed of two polynucleotide chains that coil around each other to form a double helix. The polymer carries genetic instructions for the development, functioning, growth and reproduction of al ...

*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 Prothrombin (coagulation factor II) is encoded in the human by the F2-gene. It is proteolytically cleaved during the clotting process by the prothrombinase enzyme complex to form thrombin. Thrombin (Factor IIa) (, fibrose, thrombase, throm ...

levels in a sample *"Click editors" - PCV2 Rep protein fused to nCas9 and a DNA polymerase fragment to allow for templated genome editing{{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

References

DNA-binding proteins