HIND 3 Full Form

HindIII-TypeIII Restriction Enzyme

In Hind III enzyme; 'H is refers to the Haemophilus influenzae, D is referred to the strain of Haemophilus influenzae and III refers to the sequence In which this enzyme was discovered respectively.

HindIII (Haemophilus influenzae), which is pronounced "Hin D Three," is a type II site-specific deoxyribonuclease restriction enzyme isolated from Haemophilus influenzae that hydrolyzes the cofactor Mg2+ to cleave the DNA palindromic sequence AAGCTT.

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

3'-T T C G A| A-5’

Prokaryotic organisms that utilise the restriction modification system use restriction endonucleases as defensive measures. Their job is to defend the host genome from invasion by foreign DNA, mainly bacteriophage DNA. Additionally, there is evidence that restriction enzymes may function as selfish elements alongside modification enzymes or may be engaged in genetic recombination and transposition.

Enzymes produce two ends while digesting DNA. DNA ends are the characteristics of the endpoints of linear DNA molecules, which are classified as sticky and blunt in molecular biology depending on the configuration of the complementary strands at the terminal. In sticky ends, one strand is longer than the other (usually by a few nucleotides or more), resulting in bases on the longer strand that are not linked. There are no unpaired bases on either strand since both strands in a blunt end are the same length and end at the same base position.

Structure of HindIII

The homodimer that makes up HindIII's complicated structure. It is thought to share a structural core with another type II restriction endonucleases that consist of four -sheets and a single -helix. Each component has a calculated molecular mass of 34,950 Da and comprises 300 amino acids. Despite the significance of this enzyme for molecular biology and DNA technology, little is known about how it recognizes DNA and breaks phosphodiester bonds. Though it is thought that HindIII uses a similar method of DNA identification and catalysis to another type II enzymes like EcoRI (restriction endonuclease). The amino acid sequence motif PD-(D/E)XK is present in these enzymes to coordinate Mg2+, a cation necessary for most type II restriction endonucleases to cut DNA.

Among other cations, the cofactor Mg2+ is thought to bind water molecules and transport them to the enzymes' catalytic sites. HindIII is distinctive in that it has little to no catalytic activity when Mg2+ is substituted with other cofactors, such as Mn2+, unlike the majority of known type II restriction endonucleases.

Working Mechanism of HindIII

Restriction enzymes must first attach non-specifically to the DNA backbone before localising to the restriction site, even though they cleave at specific DNA sequences. In general, the recognition sequence's bases and the restriction enzyme will establish 15 to 20 hydrogen bonds. This bonding enables a conformational shift in the DNA-enzyme complex that activates the catalytic sites through other van-der-Waals contacts.

The following hypothesised catalytic process has been developed as a result of a site-mutagenesis study in conjunction with more thorough research on metal ion-mediated catalysis in EcoRV, despite the absence of data supporting an accurate mechanism for the cleavage of DNA by HindIII.

Asp-90's carboxylate is thought to stabilise the departing hydroxide anion through the coordination of Mg2+, whereas Lys-92's catalytic residue stabilises and orients the attacking water nucleophile during the hydrolysis of DNA by EcoRV. Furthermore, the Asp-74 residue must be in the proper location for enzymatic activity, suggesting that it contributes to the attacking water molecule's increased nucleophilicity. It is therefore postulated that Lys-125, Asp-123, and Asp-108 of HindIII act similarly to Lys-92, Asp-90, and Asp-74 in EcoRV, respectively, as a result of the site-mutagenesis experiments previously described. Asp-108 increases the nucleophilicity of the attacking water molecule while Lys-125 places it. Asp-123 binds to Mg2+, stabilising the existing hydroxide ion in the process.

Restriction Enzyme

An enzyme known as a restriction enzyme, also known as a restriction endonuclease, restriction enzyme, REase, ENase, or restrictase, cleaves DNA into pieces at or near particular recognition sites inside molecules known as restriction sites. One subset of the larger endonuclease category of enzymes is restriction enzymes. The four different types of restriction enzymes are often categorised according to their structure and whether they cleave the DNA substrate at the recognition site or at a distant location. All restriction enzymes split the DNA double helix into two pieces, one through each strand's sugar-phosphate backbone.

Types of Restriction Enzymes

Recognized restriction enzymes fall into one of four categories: I, II, III, or IV. These categories are distinguished principally by their structure, cleavage site, specificity, and cofactors. In contrast to the type II system, where the restriction enzyme is independent of its methylase, types I and III enzymes are comparable in that both restriction and methylase actions are carried out by one sizable enzyme complex. The fact that type II restriction enzymes cleave DNA at particular locations inside the recognition site as opposed to types I and III's random cleavage of DNA, often hundreds of bases from the recognition sequence, is another way that they differ from types I and III. Many different bacterial species have produced thousands of type II restriction enzymes. A few hundred different sequences, typically four to eight bases long, are recognized by these enzymes. Only methylated DNA is broken down by type IV restriction enzymes, which also exhibit poor sequence selectivity.