HindIII

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HindIII restriction endonuclease
2e52.png
Crystallographic structure of the HindIII restriction endonuclease dimer (cyan and green) complexed with double helical DNA (brown) based on the PDB: 2E52 ​ coordinates.
Identifiers
SymbolRE_Hindiii
Pfam PF09518
InterPro IPR019043
hindIIIR type II restriction endonuclease
Identifiers
SymbolhindIIIR
NCBI gene 950303
PDB 2e52 More structures
UniProt P43870
Other data
EC number 3.1.21.4

HindIII (pronounced "Hin D Three") is a type II site-specific deoxyribonuclease restriction enzyme isolated from Haemophilus influenzae that cleaves the DNA palindromic sequence AAGCTT in the presence of the cofactor Mg2+ via hydrolysis. [1]

Contents

HindIII restrictions process results in formation of overhanging palindromic sticky ends. HindIII Restriction site and sticky ends vector.svg
HindIII restrictions process results in formation of overhanging palindromic sticky ends.

The cleavage of this sequence between the AA's results in 5' overhangs on the DNA called sticky ends:

5'-A |A G C T T-3'

3'-T T C G A| A-5'

Restriction endonucleases are used as defense mechanisms in prokaryotic organisms in the restriction modification system. Their primary function is to protect the host genome against invasion by foreign DNA, primarily bacteriophage DNA. There is also evidence that suggests the restriction enzymes may act alongside modification enzymes as selfish elements, or may be involved in genetic recombination and transposition. [2]

Enzyme Structure

BglII catalytic site, showing the coordination of Asp 84 and Mg with water BglII catalytic site 2.png
BglII catalytic site, showing the coordination of Asp 84 and Mg with water

The structure of HindIII is complex, and consists of a homodimer. Like other type II restriction endonucleases, it is believed to contain a common structural core comprising four β-sheets and a single α-helix. Each subunit contains 300 amino acids and the predicted molecular mass is 34,950 Da. Despite the importance of this enzyme in molecular biology and DNA technology, little information is available concerning the mechanism of DNA recognition and phosphodiester bond cleavage. [1] However, it is believed that HindIII utilizes a common mechanism of recognition and catalysis of DNA found in other type II enzymes such as EcoRI, BamHI, and BglII. These enzymes contain the amino acid sequence motif PD-(D/E)XK to coordinate Mg2+, a cation required to cleave DNA in most type II restriction endonucleases. [4] The cofactor Mg2+ is believed to bind water molecules and carry them to the catalytic sites of the enzymes, among other cations. Unlike most documented type II restriction endonucleases, HindIII is unique in that it has little to no catalytic activity when Mg2+ is substituted for other cofactors, such as Mn2+. [1]

Site-directed mutagenesis

Despite the uncertainty concerning the structure-catalysis relationship of type II endonucleases, site-directed mutagenesis of the restriction endonuclease HindIII has provided much insight into the key amino acid residues involved. In particular, substitutions of Asn for Lys at residue 125 and Leu for Asp at residue 108 significantly decreased DNA binding and the catalytic function of HindIII. [1] In a separate mutagenesis study it was shown that a mutation at residue 123 from Asp to Asn reduced enzymatic activity. Despite the fact that this residue is most likely responsible for the unwinding of DNA and coordination to water rather than direct interaction with the attacking nucleophile, its specific function is unknown. [4]

Proposed mechanism

While restriction enzymes cleave at specific DNA sequences, they are first required to bind non-specifically with the DNA backbone before localizing to the restriction site. On average, the restriction enzyme will form 15-20 hydrogen bonds with the bases of the recognition sequence. With the aid of other van der Waals interactions, this bonding facilitates a conformational change of the DNA-enzyme complex which leads to the activation of catalytic centers. [2]

Despite the lack of evidence suggesting an exact mechanism for the cleavage of DNA by HindIII, site-mutagenesis analysis coupled with more detailed studies of metal ion-mediated catalysis in EcoRV have led to the following proposed catalytic mechanism. It has been suggested that during the hydrolysis of DNA by EcoRV the catalytic residue Lys-92 stabilizes and orients the attacking water nucleophile, while the carboxylate of Asp-90 stabilizes the leaving hydroxide anion through to coordination of Mg2+. Furthermore, enzymatic function is dependent upon the correct position of the Asp-74 residue, suggesting has a role in increasing the nucleophilicity of the attacking water molecule. [5]

As a result of the site-mutagenesis experiments previously outlined, it is thus proposed that Lys-125, Asp-123, and Asp-108 of HindIII function similarly to Lys-92, Asp-90, and Asp-74 in EcoRV, respectively. Lys-125 positions the attacking water molecule while Asp-108 improves its nucleophilicity. Asp-123 coordinates to Mg2+ which in turn stabilizes the leaving hydroxide ion.

Uses in research

HindIII as well as other type II restriction endonucleases are very useful in modern science, particularly in DNA sequencing and mapping. Unlike type I restriction enzymes, type II restriction endonucleases perform very specific cleaving of DNA. Type I restriction enzymes recognize specific sequences, but cleave DNA randomly at sites other than their recognition site whereas type II restriction enzymes cleave only at their specific recognition site. [6] Since their discovery in the early 1970s, type II restriction enzymes have revolutionized the way scientists work with DNA, particularly in genetic engineering and molecular biology.

Major uses of type II restriction enzymes include gene analysis and cloning. They have proven to be ideal modeling systems for the study of protein-nucleic acid interactions, structure-function relationships, and the mechanism of evolution. [2] They make good assays for the study of genetic mutations by their ability to specifically cleave DNA to allow the removal or insertion of DNA. Through the use of restriction enzymes, scientists are able to modify, insert, or remove specific genes, a very powerful tool especially when it comes to modifying an organism's genome.

Related Research Articles

A restriction enzyme, restriction endonuclease, or restrictase is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class of the broader endonuclease group of enzymes. Restriction enzymes are commonly classified into five types, which differ in their structure and whether they cut their DNA substrate at their recognition site, or if the recognition and cleavage sites are separate from one another. To cut DNA, all restriction enzymes make two incisions, once through each sugar-phosphate backbone of the DNA double helix.

Protein engineering is the process of developing useful or valuable proteins. It is a young discipline, with much research taking place into the understanding of protein folding and recognition for protein design principles. It is also a product and services market, with an estimated value of $168 billion by 2017.

DNA methyltransferase Class of enzymes

In biochemistry, the DNA methyltransferase family of enzymes catalyze the transfer of a methyl group to DNA. DNA methylation serves a wide variety of biological functions. All the known DNA methyltransferases use S-adenosyl methionine (SAM) as the methyl donor.

Ribonuclease Class of enzyme that catalyzes the degradation of RNA

Ribonuclease is a type of nuclease that catalyzes the degradation of RNA into smaller components. Ribonucleases can be divided into endoribonucleases and exoribonucleases, and comprise several sub-classes within the EC 2.7 and 3.1 classes of enzymes.

Nuclease

A nuclease is an enzyme capable of cleaving the phosphodiester bonds between nucleotides of nucleic acids. Nucleases variously effect single and double stranded breaks in their target molecules. In living organisms, they are essential machinery for many aspects of DNA repair. Defects in certain nucleases can cause genetic instability or immunodeficiency. Nucleases are also extensively used in molecular cloning.

Aspartate transaminase Class of enzymes

Aspartate transaminase (AST) or aspartate aminotransferase, also known as AspAT/ASAT/AAT or (serum) glutamic oxaloacetic transaminase, is a pyridoxal phosphate (PLP)-dependent transaminase enzyme that was first described by Arthur Karmen and colleagues in 1954. AST catalyzes the reversible transfer of an α-amino group between aspartate and glutamate and, as such, is an important enzyme in amino acid metabolism. AST is found in the liver, heart, skeletal muscle, kidneys, brain, and red blood cells. Serum AST level, serum ALT level, and their ratio are commonly measured clinically as biomarkers for liver health. The tests are part of blood panels.

Endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain. Some, such as deoxyribonuclease I, cut DNA relatively nonspecifically, while many, typically called restriction endonucleases or restriction enzymes, cleave only at very specific nucleotide sequences. Endonucleases differ from exonucleases, which cleave the ends of recognition sequences instead of the middle (endo) portion. Some enzymes known as "exo-endonucleases", however, are not limited to either nuclease function, displaying qualities that are both endo- and exo-like. Evidence suggests that endonuclease activity experiences a lag compared to exonuclease activity.

AP endonuclease Enzyme involved in DNA repair

Apurinic/apyrimidinic (AP) endonuclease is an enzyme that is involved in the DNA base excision repair pathway (BER). Its main role in the repair of damaged or mismatched nucleotides in DNA is to create a nick in the phosphodiester backbone of the AP site created when DNA glycosylase removes the damaged base.

DNA adenine methylase Prokaryotic enzyme

DNA adenine methylase, (Dam methylase) is an enzyme that adds a methyl group to the adenine of the sequence 5'-GATC-3' in newly synthesized DNA. Immediately after DNA synthesis, the daughter strand remains unmethylated for a short time. It is an orphan methyltransferase that is not part of a restriction-modification system and regulates gene expression. This enzyme catalyses the following chemical reaction

<i>Eco</i>RV

EcoRV is a type II restriction endonuclease isolated from certain strains of Escherichia coli. It has the alternative name Eco32I.

<i>Bam</i>HI

BamHI is a type II restriction endonuclease, having the capacity for recognizing short sequences of DNA and specifically cleaving them at a target site. This exhibit focuses on the structure-function relations of BamHI as described by Newman, et al. (1995). BamHI binds at the recognition sequence 5'-GGATCC-3', and cleaves these sequences just after the 5'-guanine on each strand. This cleavage results in sticky ends which are 4 bp long. In its unbound form, BamHI displays a central b sheet, which resides in between α-helices.

Homing endonuclease

The homing endonucleases are a collection of endonucleases encoded either as freestanding genes within introns, as fusions with host proteins, or as self-splicing inteins. They catalyze the hydrolysis of genomic DNA within the cells that synthesize them, but do so at very few, or even singular, locations. Repair of the hydrolyzed DNA by the host cell frequently results in the gene encoding the homing endonuclease having been copied into the cleavage site, hence the term 'homing' to describe the movement of these genes. Homing endonucleases can thereby transmit their genes horizontally within a host population, increasing their allele frequency at greater than Mendelian rates.

Nuclease S1

Nuclease S1 is an endonuclease enzyme that splits single-stranded DNA (ssDNA) and RNA into oligo- or mononucleotides. This enzyme catalyses the following chemical reaction

<i>Bgl</i>II

BglII is a type II restriction endonuclease isolated from certain strains of Bacillus globigii.

R.EcoRII Restriction enzyme

Restriction endonuclease (REase) EcoRII is an enzyme of restriction modification system (RM) naturally found in Escherichia coli, a Gram-negative bacteria. Its molecular mass is 45.2 kDa, being composed of 402 amino acids.

PstI is a type II restriction endonuclease isolated from the Gram negative species, Providencia stuartii.

<i>Eco</i>RI

EcoRI is a restriction endonuclease enzyme isolated from species E. coli. It is a restriction enzyme that cleaves DNA double helices into fragments at specific sites, and is also a part of the restriction modification system. The Eco part of the enzyme's name originates from the species from which it was isolated - "E" denotes generic name which is "Escherichia" and "co" denotes species name, "coli" - while the R represents the particular strain, in this case RY13, and the I denotes that it was the first enzyme isolated from this strain.

References

  1. 1 2 3 4 Tang, D; et al. (2000). "Mutational analyses of restriction endonuclease-HindIII mutant E86K with higher activity and altered specificity". Protein Engineering. 13 (4): 283–9. doi: 10.1093/protein/13.4.283 . PMID   10810160.
  2. 1 2 3 Pingoud, Alfred; Jeltsch, Albert. (2001). "Structure and function of type II restriction endonucleases". Nucleic Acids Research. 29 (18): 3705–27. doi:10.1093/nar/29.18.3705. PMC   55916 . PMID   11557805.
  3. ↑ Lukacs C, et al. (2000). "Understanding the immutability of restriction enzymes: crystal structure of BglII and its DNA substrate at 1.5 A resolution". Nat. Struct. Biol. 7 (2): 134–40. doi:10.1038/72405. PMID   10655616. S2CID   20478739.
  4. 1 2 Tang D, et al. (1999). "Site-directed mutagenesis of restriction endonuclease HindIII". Biosci. Biotechnol. Biochem. 63 (10): 1703–7. doi: 10.1271/bbb.63.1703 . PMID   10586498.[ permanent dead link ]
  5. ↑ Horton N, Newberry K, Perona J (1999). "Metal ion-mediated substrate-assisted catalysis in type II restriction endonucleases". Proc. Natl. Acad. Sci. USA. 95 (23): 13489–94. doi:10.1073/pnas.95.23.13489. PMC   24846 . PMID   9811827.
  6. ↑ Roberts, Richard J. (2005). "How restriction enzymes became the workhorses of molecular biology". Proc. Natl. Acad. Sci. USA. 102 (17): 5905–8. doi:10.1073/pnas.0500923102. PMC   1087929 . PMID   15840723.
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