Home  Contacts  Order  Catalog  Support
 Search  Alphabetical Index  Numerical Index
Fermentas logo
 Restriction Enzymes  Modifying Enzymes  PCR, qPCR, RT-PCR & dNTPs  Molecular Cloning  Nucleic Acid Purification
 Molecular Labeling & Detection  In vitro Transcription  Electrophoresis Products  Nucleotides  Transfection Reagents  Reagents
S U P P O R T
 

Introduction. Classification of Restriction Enzymes

Restriction Enzymes

Restriction enzymes, due to their high specificity and ease of use, are important tools in studies of DNA primary structure, recombinant DNA technology and other fields of molecular genetics and molecular biology. More than 3500 type II restriction enzymes, exhibiting 241 different specificities, have been isolated. Since 1977, the launch of its screening program, Fermentas has discovered about 30% of all restriction enzymes described currently in the Restriction Enzyme database (REBASE), and is now a leading global manufacturer of restriction enzymes, offering 175 commercial restriction enzymes.

Active restriction enzyme screening uncovers new specificities, making Fermentas the supplier of choice for not only commonly used restriction enzymes, but also new, unique enzymes not supplied by other companies.

In-depth knowledge of restriction enzymes, gained over the past 30 years, particularly structure-activity relationships at the molecular level has led to a major breakthrough. Fermentas scientists have created two "artificial" specific enzymes - Eco57MI restriction enzyme and Nb.Bpu10I site and strand specific nicking enzyme, using patent-pending genetic engineering methods.

Although the phenomenon of host specificity was initially observed by Luria and Human in the early 1950s (1), it was nearly a decade later that Arber and Dussoix predicted its molecular basis (2). They proposed that host specificity was based on a two-enzyme system: a restriction enzyme which recognizes specific DNA sequences and is able to cleave the foreign invading DNA upon entering the bacterial cell, and a modification enzyme (methylase) responsible for protecting host DNA against the action of its own restriction enzyme. Restriction enzyme and modification methylase were thought to recognize the same nucleotide sequence and together form a restriction-modification (R-M) system. In 1968, restriction-modification enzymes EcoB and EcoK (3, 4) were isolated and classified as type I enzymes. Since they cleave DNA at random positions, they can not serve as tools to excise specific fragments. Two years later Smith and Wilcox (5) isolated and characterized the first type II restriction enzyme, HindII, that cleaved DNA in well-defined fragments. This discovery revolutionized research into gene structure and gene expression.

R-M systems were classified into three types (I, II and III) based on the complexity of their structure, cofactor requirements and substrate specificity (6). Most characterized enzymes belong to the type II. As the number of restriction enzymes increases, more and more of identified enzymes do not fit into any of these three classes. Due to their unusual properties, such enzymes have been classified as new kinds of restriction enzymes: type IIS, type IV, Bcg-like, type 1½ (7-10). Recently the new unified classification of restriction enzymes has been suggested (11).

Type II R-M Systems

Type II R-M systems are simple and the best-studied. Restriction enzymes are usually homodimers that coexist in bacterial cells with a separate methylase protein. The restriction enzymes require only Mg2+ for activity and cleave DNA within the recognition sites, leaving 5'-P and 3'-OH termini. The corresponding modification enzymes require only S-adenosylmethionine as a cofactor and methylate both strands of DNA at a specific base resulting in N6-methyladenine, 5-methylcytosine or N4-methylcytosine (12, 13). Type II R-M system enzymes recognize nucleotide palindromes 4-8 bp in length, interrupted palindromes with some unspecific nucleotides between flanking nucleotides or partially palindromic sequences with ambiguous nucleotides at certain positions. For most nucleotide sequences, more than one enzyme is available that recognizes that sequence. According to the nomenclature of restriction enzymes, first discovered restriction enzymes possessing a unique specificity are called prototypes. Subsequently discovered enzymes with the specificity of the prototype are called isoschizomers. Depending on the position of cleavage, restriction enzymes produce "sticky" (with 5'- or 3'-overhang) or "blunt" ends. Restriction enzymes recognizing the same nucleotide sequence but having different cleavage specificity are called neoschizomers. Different experimental goals may dictate the use of a different isoschizomer.

Type IIS Enzymes

Type IIS enzymes, which recognize asymmetric base sequences and cleave DNA at a specified position up to 20 base pairs outside of the recognition site, are of the special interest. Blunting the ends of their digestion products, followed by ligation, does not destroy their recognition sites. This property is useful in several applications, including the generation of deletions of increasing length (14) and mapping the sequence specificity of DNA modification (15) and PCR product cloning. Due to the asymmetric nature of their recognition sequences, type IIS R-M systems comprise two methylases, one for each strand, sometimes each methylating a different base (16).

Type II restriction-modification systems are widespread among bacteria, but have also been isolated from phages, archeabacteria and viruses of eukaryotic algae.

References

  1. Luria, S.E., Human, M.L., A nonhereditary, host-induced variation of bacterial viruses, J. Bacteriol., 64, 557-569, 1952.

  2. Arber, W., Dussoix, D., Host specificity of DNA producted by Escherichia coli: I. Host controlled modification of bacteriophage lambda, J. Mol. Biol., 5, 18-36, 1962.
  3. Linn, S., Arber, S., Host specificity of DNA produced by Escherichia coli, X. In vitro restriction of phage fd replicative form, Proc. Natl. Acad. Sci USA, 59, 1300-1306, 1968.
  4. Meselson, M., Yuan, R., DNA restriction enzyme from E.coli, Nature, 217, 1110-1114, 1968.
  5. Smith, H.O., Wilcox, K.W., A restriction enzyme from Hemophilus influenzae. I. Purification and general properties, J. Mol. Biol., 51, 379-391, 1970.
  6. Yuan, R., Structure and mechanism of multifunctional restriction endonucleases, Annu. Rev. Biochem., 150, 285-315, 1981.
  7. Szybalski, W., et al., Class-IIS restriction enzymes - a review, Gene, 100, 13-26, 1991.
  8. Janulaitis, A., et al., Purification and properties of the Eco57I restriction endonuclease and methylase-prototypes of a new class (type IV), Nucleic Acids Acids Res., 20, 6043-6049, 1992.
  9. Sears, L.E., et al., BaeI, another unusual BcgI-like restriction endonuclease, Nucleic Acids Res., 24, 3590-3592, 1996.
  10. Mernagh, D., et al., AhdI, a new class of restriction-modification system?, Biochem. Soc. Trans., 27, A126, 1999.
  11. Roberts, R.J., et al., A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes, Nucleic Acids Res., 31, 1805-1812, 2003.
  12. McClelland, M., The effect of site specific methylation on restriction endonuclease cleavage (update), Nucleic Acids Res., 11, r169-r173, 1983.
  13. Janulaitis, A., et al., Cytosine modification in DNA by BcnI methylase yields N4-methylcytosine, FEBS Lett., 161, 131-134, 1983.
  14. Hasan, N., et al., A novel multistep method for generating precise unidirectional deletions using BspMI, a class-IIS restriction enzyme, Gene, 50, 55-62, 1986.
  15. Posfai, G., Szybalski, W., A simple method for locating methylated bases in DNA, as applied to detect asymmetric methylation by M.FokIA, Gene, 69, 147-151, 1988.
  16. Bitinaite, J., et al., Alw26I, Eco31I and Esp3I - type IIs methyltransferases modifying cytosine and adenine in complementary strands of the target DNA, Nucleic Acids Res., 20, 4981-4985, 1992.
 Home  Search  Contacts  Order  Catalog  Support

catalog@fermentas.com

Updated lapkričio 08, 2006 11:10