# HindIII
**HindIII** is a Type II site-specific restriction endonuclease (EC 3.1.21.4) from the bacterium *Haemophilus influenzae* strain Rd that recognizes the palindromic DNA sequence 5′-AAGCTT-3′ and cleaves both strands between the first two bases, written A/AGCTT, leaving four-base 5′ overhangs (sticky ends)[1](https://www.ncbi.nlm.nih.gov/mesh?Cmd=DetailsSearch&Db=mesh&Term=%22Deoxyribonuclease+HindIII%22%5BMeSH+Terms%5D). It is one of the most widely used enzymes in molecular cloning and was among the orthodox Type II enzymes, with EcoRI and EcoRV, that defined the class[2](https://academic.oup.com/nar/article/42/1/3/2438402).
| Key fact | Detail |
|---|---|
| Recognition and cut site | A/AGCTT, cleaved at the slash; the site is palindromic and 6 bp long[1](https://www.ncbi.nlm.nih.gov/mesh?Cmd=DetailsSearch&Db=mesh&Term=%22Deoxyribonuclease+HindIII%22%5BMeSH+Terms%5D) • [3](https://www.neb.com/en-us/tools-and-resources/selection-charts/alphabetized-list-of-recognition-specificities) |
| Source organism | *Haemophilus influenzae* strain Rd; commercial enzyme is produced in an *E. coli* strain carrying the cloned HindIII gene[1](https://www.ncbi.nlm.nih.gov/mesh?Cmd=DetailsSearch&Db=mesh&Term=%22Deoxyribonuclease+HindIII%22%5BMeSH+Terms%5D) • [4](https://www.neb.com/en-ca/products/r0104-hindiii) |
| Enzyme class | Orthodox Type II restriction endonuclease; homodimer of roughly 2 × 30 kDa that requires Mg2+ and cuts within its recognition site[5](https://pmc.ncbi.nlm.nih.gov/articles/PMC55916/) • [6](https://www.neb.com/en-gb/products/restriction-endonucleases/types-of-restriction-endonucleases) |
| End structure | Sticky ends (5′ overhangs), which is why HindIII is a standard choice for directional cloning alongside enzymes such as EcoRI and BamHI[7](https://mlsu.ac.in/econtents/64_Enzymes%20used%20in%20GE.pdf) |
| Unit definition (NEB) | One unit digests 1 µg of λ DNA in 1 hour at 37 °C in a 50 µl reaction[4](https://www.neb.com/en-ca/products/r0104-hindiii) |
| Methylation sensitivity | Not sensitive to dam, dcm, or mammalian CpG methylation[4](https://www.neb.com/en-ca/products/r0104-hindiii) |
| Engineered variant | HindIII-HF (NEB #R3104), with the same specificity but significantly reduced star activity and 100% activity in rCutSmart Buffer[8](https://www.neb.com/en/products/r3104-hindiii-hf) |
## Recognition sequence and discovery
Restriction enzymes were first characterized in *H. influenzae*: Hamilton Smith and Kent Wilcox isolated and characterized endonuclease R, later renamed HindII, from extracts of strain Rd, the first restriction enzyme described[9](https://www.nature.com/articles/miledna04). The recognition sequence of the third enzyme of the same strain, R.HindIII, was reported in the *Journal of Molecular Biology* in 1975. The authors showed that the enzyme degrades foreign DNA but not homologous DNA and concluded that it recognizes and cleaves the sequence AAGCTT, giving termini bearing short cohesive ends[10](https://www.sciencedirect.com/science/article/pii/0022283675902326).
The HindIII site proved useful even in early sequence analysis of related enzymes: phage T7 DNA, the substrate used to work out the HindII recognition sequence, happens to contain no HindIII sites, which prevented HindIII cutting from interfering with that analysis[11](https://academic.oup.com/nar/article/42/12/7489/1104749).
## Structure and mechanism
HindIII belongs to the orthodox Type II enzymes, which are homodimers of about 2 × 30 kDa molecular mass that recognize palindromic sequences 4 to 8 bp long and, in the presence of Mg2+, cleave the two DNA strands within or immediately adjacent to the recognition site to yield a 5′-phosphate and a 3′-OH end[5](https://pmc.ncbi.nlm.nih.gov/articles/PMC55916/). Because the A/AGCTT cut falls at the same relative position on both strands of the palindromic site, each product end carries a single-stranded 5′ overhang of AGCT. These complementary sticky ends anneal to matching ends and are joined by [DNA ligase](https://www.edgepedia.org/dna-ligase), which is the basis of HindIII's use in cloning[7](https://mlsu.ac.in/econtents/64_Enzymes%20used%20in%20GE.pdf).
Like other Type II enzymes, HindIII requires Mg2+ for catalysis; mechanistic work across the family indicates two divalent metal binding sites per active center, a high-affinity site required for cleavage and a second site whose effect depends on which ion occupies it[12](https://pmc.ncbi.nlm.nih.gov/articles/PMC4081073/).
## Laboratory use
The commercial enzyme (NEB #R0104) is supplied from an *E. coli* strain carrying the HindIII gene from *H. influenzae* Rd (ATCC 51907)[4](https://www.neb.com/en-ca/products/r0104-hindiii). Standard reaction conditions are 1X NEBuffer r2.1 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 100 µg/ml recombinant albumin, pH 7.9) incubated at 37 °C; the enzyme is heat-inactivated at 80 °C for 20 minutes[4](https://www.neb.com/en-ca/products/r0104-hindiii). Since July 2022 the NEB formulation has been Time-Saver qualified, digesting substrate DNA in 5 to 15 minutes[4](https://www.neb.com/en-ca/products/r0104-hindiii).
Two practical qualifications apply. The enzyme shows lower activity on some supercoiled plasmids, with more than 1 unit required to digest 1 µg of plasmid DNA, and it is not sensitive to dam, dcm, or CpG methylation, so methylated DNA from common cloning hosts cuts normally[4](https://www.neb.com/en-ca/products/r0104-hindiii).
**Star activity.** Under non-recommended conditions, restriction enzymes lose fidelity and cut at sites similar to the cognate site, a behavior called star activity. HindIII is among the enzymes for which star activity has been reported[13](https://pmc.ncbi.nlm.nih.gov/articles/PMC2396408/). Conditions that promote it include high glycerol concentration (above 5% v/v), high enzyme-to-DNA ratio (usually above 100 U per µg of DNA), low ionic strength (below 25 mM salt), high pH (above 8.0), organic solvents such as DMSO or ethanol, and substitution of Mg2+ with Mn2+ or Co2+ • [13](https://pmc.ncbi.nlm.nih.gov/articles/PMC2396408/). Extended digestion is a specific caution for HindIII[4](https://www.neb.com/en-ca/products/r0104-hindiii). The engineered High Fidelity version, HindIII-HF (NEB #R3104), has the same A/AGCTT specificity but is engineered for significantly reduced star activity and full activity in a single buffer, rCutSmart[8](https://www.neb.com/en/products/r3104-hindiii-hf).
## Isoschizomers and nomenclature
The enzyme's name follows the restriction enzyme convention: HindIII is the third restriction enzyme characterized from *H. influenzae* strain Rd. Numerous isoschizomers, enzymes from other organisms with the same recognition sequence and cut position, have been identified; MeSH lists entry terms including Endonuclease EcoVIII, Endonuclease BstFI, Endonuclease BpeI, and Endonuclease HsuI, among others[1](https://www.ncbi.nlm.nih.gov/mesh?Cmd=DetailsSearch&Db=mesh&Term=%22Deoxyribonuclease+HindIII%22%5BMeSH+Terms%5D).
## References
1. [Deoxyribonuclease HindIII, MeSH, NCBI](https://www.ncbi.nlm.nih.gov/mesh?Cmd=DetailsSearch&Db=mesh&Term=%22Deoxyribonuclease+HindIII%22%5BMeSH+Terms%5D)
2. [Highlights of the DNA cutters: a short history of the restriction enzymes, Nucleic Acids Research](https://academic.oup.com/nar/article/42/1/3/2438402)
3. [Alphabetized List of Recognition Sequences, New England Biolabs](https://www.neb.com/en-us/tools-and-resources/selection-charts/alphabetized-list-of-recognition-specificities)
4. [HindIII (NEB #R0104), New England Biolabs](https://www.neb.com/en-ca/products/r0104-hindiii)
5. [Structure and function of type II restriction endonucleases, PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC55916/)
6. [Types of restriction endonucleases, New England Biolabs](https://www.neb.com/en-gb/products/restriction-endonucleases/types-of-restriction-endonucleases)
7. [Enzymes used in genetic engineering, NPTEL course material](https://mlsu.ac.in/econtents/64_Enzymes%20used%20in%20GE.pdf)
8. [HindIII-HF (NEB #R3104), New England Biolabs](https://www.neb.com/en/products/r3104-hindiii-hf)
9. [Making the cut, Nature Reviews Genetics](https://www.nature.com/articles/miledna04)
10. [Recognition sequence of restriction endonuclease III from Hemophilus influenzae, Journal of Molecular Biology](https://www.sciencedirect.com/science/article/pii/0022283675902326)
11. [Type II restriction endonucleases—a historical perspective and more, Nucleic Acids Research](https://academic.oup.com/nar/article/42/12/7489/1104749)
12. [Type II restriction endonucleases—a historical perspective and more, PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC4081073/)
13. [The Fidelity Index provides a systematic quantitation of star activity of DNA restriction endonucleases, PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC2396408/)
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*Topic: Encyclopedia › Life and health › Biological foundations › Biochemistry and metabolism › Enzyme classes and activities › DNA and RNA processing enzyme activities › Nucleases and restriction enzymes › Type II restriction enzymes and type IIS*
*Initially written Sep 17, 2026 · Reviewed: Oct 21, 2026 · Edited: Oct 15, 2026; Oct 21, 2026 · Last review: Oct 21, 2026*
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License: Edgepedia Community License 1.0, https://www.edgepedia.org/edgepedia/license