HindIII
HindIII restriction endonuclease | |||||||||
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Identifiers | |||||||||
Symbol | RE_Hindiii | ||||||||
Pfam | PF09518 | ||||||||
InterPro | IPR019043 | ||||||||
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hindIIIR type II restriction endonuclease | |||||||
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Identifiers | |||||||
Symbol | hindIIIR | ||||||
UniProt | P43870 | ||||||
Other data | |||||||
EC number | 3.1.21.4 | ||||||
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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]
The cleavage of this sequence between the AA's results in 5' overhangs on the DNA called
5'-A |A G C T T-3'
3'-T T C G A| A-5'
Restriction endonucleases are used as defense mechanisms in
Enzyme Structure
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
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
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
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