| Type-2 restriction enzyme HaeIII | |||||||
|---|---|---|---|---|---|---|---|
 The crystal structure of HaeIII enzyme convalently complexed to DNA: an extrahelical cytosine and rearranged base pairing. | |||||||
| Identifiers | |||||||
| Organism | |||||||
| Symbol | haeIIIR | ||||||
| RefSeq (Prot) | WP_006996034.1 | ||||||
| UniProt | O68584 | ||||||
| Other data | |||||||
| EC number | 3.1.21.4 | ||||||
| Chromosome | Genomic: 0.7 - 0.7 Mb | ||||||
| |||||||
HaeIII is one of many restriction enzymes (endonucleases) a type of prokaryotic DNA that protects organisms from unknown, foreign DNA.[1] It is a restriction enzyme used in molecular biology laboratories. It was the third endonuclease to be isolated from the Haemophilus aegyptius bacteria. The enzyme's recognition site—the place where it cuts DNA molecules—is the GGCC nucleotide sequence which means it cleaves DNA at the site 5′-GG/CC-3. The recognition site is usually around 4-8 bps[1].This enzyme's gene has been sequenced and cloned. This is done to make DNA fragments in blunt ends. HaeIII is not effective for single stranded DNA cleavage.
YouTube Encyclopedic
-
1/3Views:12 72933 65353 960
-
DNA restriction enzymes
-
Restriction enzymes: Definition, Types and Cut Patterns
-
Restriction digest
Transcription
The tools that are available to molecular biologists ... many of them come from molecular biology bacteria....we don't think about it but bacteria, kind of, live in a difficult world they have their own set of problems one of the problems that bacteria have, is invading DNA in the form of phages, vectors and viruses and things like that to fight off invading DNA, bacteria make restriction endonucleases these are weapons for bacteria to fight off invading DNA the restriction endonucleases recognize specific places in DNA and cut the DNA this is a defense mechanism for bacteria but it's a tool for molecular biologists let's go straight to an example we have HindIII. HindIII is produced by bacteria and if an invading DNA enters the bacteria this enzyme will break the DNA right at this spot right here another tool which is used by molecular biologists, which comes from bacteria is something called a sticky end EcoRI, the enzyme EcoRI, makes a break at a location that's a GAATTC and notice it's.. from one direction it's opposite going the other direction but.. and it makes a staggered end and makes a sticky end one of the tools in molecular biology involves taking DNA apart and stitching it back together this has been referred to as molecular scissors and molecular glue this comes from sticky ends that are produced by restriction endonucleases another tool would be in a situation for genomic sequencing they would subject the DNA to a series of restriction endonucleases ....end up with a series of fragments.... sequence those fragments and then use computers to put it all back together this is the basis of shotgun genomic sequencing another application is in the world of forensics if you take a piece of human DNA and subject it to restriction endonucleases two people will not have the same fragments some of the early, first generation, DNA fingerprinting use this principle
Properties
HaeIII has a molecular weight of 37126. After a 2-10-fold of HaeIII takes place, there is overdigestion of a DNA substrate. This results in 100% being cut, more than 50% of fragments being ligated, and more than 95% being recut. Heat inactivation comes at about 80 °C for 20 minutes. The locus of the HaeIII enzyme is on AF05137, and is linear with 957 base pairs.
History
HaeIII along with other restriction enzymes were discovered in 1970 by Werner Arber and Matthew Meselson. The HaeIII methyltransferase also known as MTase gene from Haemophilus aegyptius (recognition sequence: 5′-GGCC-3′) was made into Escherichia coli (E.coli) in the plasmid vector pBR322. The gene was extracted from a single EcoRI fragment and a single HindIII enzyme fragment. Clones carrying additional adjacent fragments were found to code for the HaeIII restriction enzyme.
Function
The enzyme cleaves the DNA at the positions where the GGCC sequence is found. The cleavage occurs between the second and the third nucleotides (G and C). The resulting DNA fragments are known as restriction fragments. HaeIII cuts both strands of DNA in the same location, yielding restriction fragments with blunt ends. Heat denaturation occurs at 80°C after 20 minutes.[2]
Methylase
Haemophilus aegyptius also carries a methylase dubbed HaeIIIM (P20589) that methylates the internal cytosines in the GGCC sequence. It protects sequences from being cut by HaeIII, and forms a restriction modification system. HaeIII enzyme comes from an E. coli strain that carries the cloned HaeIII modification gene from Haemophilus aegyptius.
References
- ^ a b "InterPro". www.ebi.ac.uk. Retrieved 2020-04-06.
- ^ "HaeIII Restriction Enzyme". theLabRat.com. 2005.
Further reading
- Rawn JD (1989). Biochemistry (International ed.). ISBN 0-89278-405-9.
- Slatko BE, Croft R, Moran LS, Wilson GG (December 1988). "Cloning and analysis of the HaeIII and HaeII methyltransferase genes". Gene. 74 (1): 45–50. doi:10.1016/0378-1119(88)90248-x. PMID 3248732.
- Blakesley RW, Dodgson JB, Nes IF, Wells RD (October 1977). "Duplex regions in "single-stranded" phiX174 DNA are cleaved by a restriction endonuclease from Haemophilus aegyptius". The Journal of Biological Chemistry. 252 (20): 7300–6. doi:10.1016/S0021-9258(19)66969-3. PMID 71298.
- Hoffman JL (July 1986). "Chromatographic analysis of the chiral and covalent instability of S-adenosyl-L-methionine". Biochemistry. 25 (15): 4444–9. doi:10.1021/bi00363a041. PMID 3530324.
This page was last edited on 14 June 2024, at 01:15