Restriction site

Not to be confused with Recognition sequence.

Restriction sites, or restriction recognition sites, are located on a DNA molecule containing specific (4-8 base pairs in length^[1]) sequences of nucleotides, which are recognized by restriction enzymes. These are generally palindromic sequences^[2] (because restriction enzymes usually bind as homodimers), and a particular restriction enzyme may cut the sequence between two nucleotides within its recognition site, or somewhere nearby.

Function

For example, the common restriction enzyme EcoRI recognizes the palindromic sequence GAATTC and cuts between the G and the A on both the top and bottom strands. This leaves an overhang (an end-portion of a DNA strand with no attached complement) known as a sticky end^[2] on each end of AATT. The overhang can then be used to ligate in (see DNA ligase) a piece of DNA with a complementary overhang (another EcoRI-cut piece, for example).

Some restriction enzymes cut DNA at a restriction site in a manner which leaves no overhang, called a blunt end.^[2] Blunt ends are much less likely to be ligated by a DNA ligase because the blunt end doesn't have the overhanging base pair that the enzyme can recognize and match with a complementary pair.^[3] Sticky ends of DNA however are more likely to successfully bind with the help of a DNA ligase because of the exposed and unpaired nucleotides. For example, a sticky end trailing with AATTG is more likely to bind with a ligase than a blunt end where both the 5' and 3' DNA strands are paired. In the case of the example the AATTG would have a complementary pair of TTAAC which would reduce the functionality of the DNA ligase enzyme.^[4]

Applications

Restriction sites can be used for multiple applications in molecular biology such as identifying restriction fragment length polymorphisms (RFLPs). Restriction sites are also important consideration to be aware of when designing plasmids.

Databases

Several databases exist for restriction sites and enzymes, of which the largest noncommercial database is REBASE.^[5][6] Recently, it has been shown that statistically significant nullomers (i.e. short absent motifs which are highly expected to exist) in virus genomes are restriction sites indicating that viruses have probably got rid of these motifs to facilitate invasion of bacterial hosts.^[7] Nullomers Database contains a comprehensive catalogue of minimal absent motifs many of which might potentially be not-yet-known restriction motifs.

See also

• List of restriction enzyme cutting sites

References

1. Russell, Peter J. (2006). iGenetics: A Mendelian Approach. Benjamin Cummings. ISBN 978-0805346664.
2. Lehninger, Albert L.; Nelson, David L.; Cox, Michael M. (2008). Principles of Biochemistry (5th ed.). New York, NY: W.H. Freeman and Company. p. 305–306. ISBN 978-0-7167-7108-1.
3. Mousavi-Khattat, Mohammad; Rafati, Adele; Gill, Pooria (5 February 2015). "Fabrication of DNA nanotubes using origami-based nanostructures with sticky ends". Journal of Nanostructure in Chemistry. 5 (2): 177–183. doi:10.1007/s40097-015-0148-z.
4. Gao, Song; Zhang, Jiannan; Miao, Tianjin; Ma, Di; Su, Ying; An, Yingfeng; Zhang, Qingrui (28 March 2015). "A Simple and Convenient Sticky/Blunt-End Ligation Method for Fusion Gene Construction". Biochemical Genetics. 53 (1–3): 42–48. doi:10.1007/s10528-015-9669-x. PMID 25820211. S2CID 16709792.
5. Roberts, Richard J.; Vincze, Tamas; Posfai, Janos; Macelis, Dana (2009-10-21). "REBASE—a database for DNA restriction and modification: enzymes, genes and genomes". Nucleic Acids Research. 38 (suppl_1): D234–D236. doi:10.1093/nar/gkp874. ISSN 0305-1048. PMC 2808884. PMID 19846593.
6. Roberts, Richard J.; Vincze, Tamas; Posfai, Janos; Macelis, Dana (2014-11-05). "REBASE—a database for DNA restriction and modification: enzymes, genes and genomes". Nucleic Acids Research. 43 (D1): D298–D299. doi:10.1093/nar/gku1046. ISSN 1362-4962. PMC 4383893. PMID 25378308.
7. Koulouras, Grigorios; Frith, Martin C (2021-04-06). "Significant non-existence of sequences in genomes and proteomes". Nucleic Acids Research. 49 (6): 3139–3155. doi:10.1093/nar/gkab139. ISSN 0305-1048. PMC 8034619. PMID 33693858.