Restriction enzyme bound to DNA

A restriction enzyme (or restriction endonuclease) is an enzyme that cuts double-stranded DNA following its specific recognition of short nucleotide sequences, known as restriction sites, in the DNA. Endonucleases are Enzymes that cleave the Phosphodiester bond within a Polynucleotide chain in contrast to Exonucleases which cleave Phosphodiester Enzymes are Biomolecules that catalyze ( ie increase the rates of Chemical reactions Almost all enzymes are Proteins Deoxyribonucleic acid ( DNA) is a Nucleic acid that contains the genetic instructions used in the development and functioning of all known Nucleotides are Organic compounds that consist of three joined structures a nitrogenous base a Sugar, and a Phosphate group Restriction sites, or restriction recognition sites, are specific sequences of Nucleotides that are recognized by Restriction enzymes The sites are generally Such enzymes, found in bacteria and archaea, are thought to have evolved to provide a defense mechanism against invading viruses. The Bacteria ( singular: bacterium) are a large group of unicellular Microorganisms Typically a few Micrometres in length bacteria have A virus (from the Latin virus meaning Toxin or Poison) is a sub-microscopic infectious agent that is unable ^[1][2] Inside a bacterial host, the restriction enzymes selectively cut up foreign DNA in a process called restriction; host DNA is methylated by a modification enzyme (a methylase) to protect it from the restriction enzyme’s activity. Methylation is a term used in the chemical sciences to denote the attachment or substitution of a methyl group on various substrates. A methylase is an Enzyme that attaches a Methyl group to a Molecule. Collectively, these two processes form the restriction modification system. The restriction modification system ( RM system) is used by Bacteria, and perhaps other prokaryotic organisms to protect themselves from foreign DNA ^[3] To cut the DNA, a restriction enzyme makes two incisions, once through each sugar-phosphate backbone (i. e. each strand) of the DNA double helix. ^[4][5][6]

The Nobel Prize in Medicine was awarded, in 1978, to Daniel Nathans, Werner Arber, and Hamilton Smith for the discovery of restriction endonucleases. The Nobel Prize (Nobelpriset (Nobelprisen is a Swedish prize established in the 1895 will of Swedish chemist Alfred Nobel; it was first awarded in Peace, Literature Daniel Nathans ( October 30, 1928 &ndash November 16, 1999) was an American Microbiologist. Werner Arber (born June 3, 1929) is a Swiss Microbiologist and Geneticist. Hamilton Othanel Smith (born August 23, 1931) is an American Microbiologist and Nobel laureate ^[7] Their discovery lead to the development of recombinant DNA technology that allowed, for example, the large scale production of human insulin for diabetics using E. coli bacteria. Recombinant DNA is a form of synthetic DNA that is engineered through the combination or insertion of one or more DNA strands thereby combining DNA sequences Insulin is a Hormone with intensive effects on both metabolism and several other body systems (eg vascular compliance Diabetes mellitus (ˌdaɪəˈbiːtiːz or /ˌdaɪəˈbiːtəs/ /məˈlaɪtəs/ or /ˈmɛlətəs/ often referred to simply as diabetes ( Ancient Greek: grc ^[8] Over 100 restriction enzymes have since been purified and characterized from different types and strains of bacteria, and are routinely used for DNA modification and manipulation in laboratories.

Restriction enzymes as tools

See the main article on restriction digests. A restriction digest is a procedure used in Molecular biology to prepare DNA for analysis or other processing

EcoRI digestion produces "sticky" ends.

SmaI restriction enzyme cleavage produces "blunt" ends.

Recognition sequences typically are only four to twelve nucleotides long. Because there are only so many ways to arrange the four nucleotides--A,C,G and T--into a four or eight or twelve nucleotide sequence, recognition sequences tend to "crop up" by chance in any long sequence. Furthermore, restriction enzymes specific to hundreds of distinct sequences have been identified and synthesized for sale to laboratories. As a result, potential "restriction sites" appear in almost any gene or chromosome. History See also History of genetics The existence of genes was first suggested by Gregor Mendel (1822-1884 who in the 1860s studied inheritance A chromosome is an organized structure of DNA and Protein that is found in cells. Meanwhile, the sequences of some artificial plasmids include a "linker" that contains dozens of restriction enzyme recognition sequences within a very short segment of DNA. A plasmid is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA So no matter the context in which a gene naturally appears, there is probably a pair of restriction enzymes that can snip it out, and which will produce ends that enable the gene to be spliced into a "plasmid" (i. A plasmid is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA e. , which will enable what molecular biologists call "cloning" of the gene). Cloning in Biology is the process of producing populations of genetically-identical individuals that occurs in nature when organisms such as Bacteria, Insects ^[9][10]

Another use of restriction enzymes can be to find specific SNPs. A single nucleotide polymorphism ( SNP, pronounced snip) is a DNA sequence variation occurring when a single Nucleotide - A, T ^[11][12] If a restriction enzyme can be found such that it cuts only one possible allele of a section of DNA (that is, the alternate nucleotide of the SNP causes the restriction site to no longer exist within the section of DNA), this restriction enzyme can be used to genotype the sample without completely sequencing it. An allele (ˈæliːl (UK /əˈliːl/ (US (from the Greek αλληλος allelos, meaning each other) is one member of a pair or series of different forms Restriction sites, or restriction recognition sites, are specific sequences of Nucleotides that are recognized by Restriction enzymes The sites are generally The genotype is the genetic constitution of a cell an organism or an individual (i The sample is first run in a restriction digest to cut the DNA, then gel electrophoresis is performed on this digest. A restriction digest is a procedure used in Molecular biology to prepare DNA for analysis or other processing If the sample is homozygous for the common allele, the result will be two bands of DNA, because the cut will have occurred at the restriction site. Zygosity refers to the genetic condition of a Zygote. In genetics zygosity describes the similarity or dissimilarity of DNA between Homologous If the sample is homozygous for the rarer allele, the sample will show only one band, because it will not have been cut. If the sample is heterozygous at that SNP, there will be three bands of DNA. Zygosity refers to the genetic condition of a Zygote. In genetics zygosity describes the similarity or dissimilarity of DNA between Homologous This is an example of restriction mapping, see the article on restriction maps

Recognition sites

Restriction enzymes recognize a specific sequence of nucleotides^[5] and produce a double stranded cut in the DNA that prevents the phage from replicating. A restriction map is a map of known Restriction sites within a sequence of DNA. While recognition sequences vary widely, with lengths between 4 and 8 nucleotides, many of them are palindromic; that is, the sequence on one strand reads the same in the reverse direction on the complementary strand. A palindrome is a word phrase number or other sequence of units that can be read the same way in either direction (the adjustment of punctuation and spaces between words ^[13] The meaning of "palindromic" in this context is different from what one might expect from its linguistic usage: GTAATG is not a palindromic DNA sequence, but GTATAC is (GTATAC is complementary to CATATG):

5'-GTATAC-3'
   ||||||
3'-CATATG-5'

Bacteria prevent their own DNA from being cut by modifying their nucleotides via methylation. Methylation is a term used in the chemical sciences to denote the attachment or substitution of a methyl group on various substrates. ^[1]

Nomenclature

Since their discovery in the 1970s, more than 100 different restriction enzymes have been identified in different bacteria. Each enzyme is named after the bacterium from which it was isolated using a naming system based on bacterial genus, species and strain. A genus (plural genera from Γένος Latin genus "descent family type gender" is a low-level Taxonomic In Biology, a species is one of the basic units of Biological classification and a Taxonomic rank. In biology strain is a low-level Taxonomic rank used in three related ways ^[14][15] For example, the name of the EcoRI restriction enzyme was derived as shown in the box to the right. ECOR1_Crystal_Structurershpng|thumb|EcoRI crystal structure]] Eco RI (pronounced "eco R one" is a Nuclease enzyme isolated from certain strains of

Enzyme Mechanisms

There are three groups of restriction enzyme that vary in the way recognise their restriction sites and where they cut the DNA:^[16][17]

• Type I restriction enzymes cut DNA about 100 nucleotides after the recognition site and requires ATP. Adenosine-5'-triphosphate ( ATP) is a multifunctional Nucleotide that is most important as a " molecular currency" of intracellular Energy ^[18]
• Type II restriction enzymes cut DNA at the recognition site or near the recognition site (Type IIS) and for this reason are most often used in scientific experimentation. ^[13]
• Type III restriction enzymes cut DNA about 20-30 base pairs after the recognition site and requires ATP. ^[19]

Splicing together cleaved DNA fragments

The chemical bonds cleaved by restriction enzymes can be reformed by other enzymes known as DNA ligases, allowing restriction fragments carved from different chromosomes or genes to be spliced together, provided their ends are complementary (more below). A chemical bond is the physical process responsible for the attractive interactions between Atoms and Molecules and which confers stability to diatomic and polyatomic In Molecular biology, DNA ligase is a special type of Ligase ( that can link together two DNA strands that have single-strand breaks (a break in both complementary A chromosome is an organized structure of DNA and Protein that is found in cells. History See also History of genetics The existence of genes was first suggested by Gregor Mendel (1822-1884 who in the 1860s studied inheritance In Molecular biology, splicing is a modification of an RNA after transcription, in which Introns are removed and Exons are joined Many of the procedures of molecular biology and genetic engineering rely on restriction enzymes. Molecular biology is the study of Biology at a molecular level Genetic engineering, Recombinant DNA technology, genetic modification/manipulation (GM and gene splicing are terms that apply to the direct ^[20][21][22]

Recognition sequences in DNA differ for each restriction enzyme, producing differences in the length, sequence and strand orientation (5' end or the 3' end) of a sticky-end "overhang" of an enzyme restriction. Directionality, in Molecular biology, refers to the end-to-end chemical orientation of a single strand of Nucleic acid. Directionality, in Molecular biology, refers to the end-to-end chemical orientation of a single strand of Nucleic acid. DNA end or sticky end refers to the properties of the end of a Molecule of DNA.

Examples

Examples of restriction enzymes include:^[23]

5'GAATTC
3'CTTAAG

5'---G     AATTC---3'
3'---CTTAA     G---5'

5'CCWGG
3'GGWCC

5'---     CCWGG---3'
3'---GGWCC     ---5'

5'GGATCC
3'CCTAGG

5'---G     GATCC---3'
3'---CCTAG     G---5'

5'AAGCTT
3'TTCGAA

5'---A     AGCTT---3'
3'---TTCGA     A---5'

5'TCGA
3'AGCT

5'---T   CGA---3'
3'---AGC   T---5'

5'GCGGCCGC
3'CGCCGGCG

5'---GC   GGCCGC---3'
3'---CGCCGG   CG---5'

5'GANTC
3'CTNAG

5'---G   ANTC---3'
3'---CTNA   G---5'

5'GATC
3'CTAG

5'---     GATC---3'
3'---CTAG     ---3'

5'CAGCTG
3'GTCGAC

5'---CAG  CTG---3'
3'---GTC  GAC---5'

5'CCCGGG
3'GGGCCC

5'---CCC  GGG---3'
3'---GGG  CCC---5'

5'GGCC
3'CCGG

5'---GG  CC---3'
3'---CC  GG---5'

5'AGCT
3'TCGA

5'---AG  CT---3'
3'---TC  GA---5'

5'GATATC
3'CTATAG

5'---GAT  ATC---3'
3'---CTA  TAG---5'

5'GGTACC
3'CCATGG

5'---GGTAC  C---3'
3'---C  CATGG---5'

5'CTGCAG
3'GACGTC

5'---CTGCA  G---3'
3'---G  ACGTC---5'

5'GAGCTC
3'CTCGAG

5'---GAGCT  C---3'
3'---C  TCGAG---5'

5'GTCGAC
3'CAGCTG

5'---G  TCGAC---3'
3'---CAGCT  G---5'

5'AGTACT
3'TCATGA

5'---AGT  ACT---3'
3'---TCA  TGA---5'

5'GCATGC
3'CGTACG

5'---G  CATGC---3'
3'---CGTAC  G---5'

5'AGGCCT
3'TCCGGA

5'---AGG  CCT---3'
3'---TCC  GGA---5'

5'TCTAGA
3'AGATCT

5'---T  CTAGA---3'
3'---AGATC  T---5'

See also

• Star activity

References

External links

General:

• MeSH DNA Restriction Enzymes
• Firman K (2007-11-24). Medical Subject Headings ( MeSH) is a huge Controlled vocabulary (or metadata system for the purpose of indexing journal articles and books Year 2007 ( MMVII) was a Common year starting on Monday of the Gregorian calendar in the 21st century. Events 380 - Theodosius I makes his adventus, or formal Type I Restriction-Modification. University of Portsmouth. Retrieved on 2008-06-06. 2008 ( MMVIII) is the current year in accordance with the Gregorian calendar, a Leap year that started on Tuesday of the Common Events 1508 - Maximilian I Holy Roman Emperor, is defeated in Friulia by Venetian forces; he is forced to sign a three-year
• Goodsell DS (2000-08-01). 2000 ( MM) was a Leap year that started on Saturday of the Common Era, in accordance with the Gregorian calendar. Events 30 BC - Octavian (later known as Augustus enters Alexandria, Egypt, bringing it under the control of the Roman Restriction Enzymes. Molecule of the Month. RCSB Protein Data Bank. Retrieved on 2008-06-06. 2008 ( MMVIII) is the current year in accordance with the Gregorian calendar, a Leap year that started on Tuesday of the Common Events 1508 - Maximilian I Holy Roman Emperor, is defeated in Friulia by Venetian forces; he is forced to sign a three-year
• Simmer M, Secko D (2003-08-01). Year 2003 ( MMIII) was a Common year starting on Wednesday of the Gregorian calendar. Events 30 BC - Octavian (later known as Augustus enters Alexandria, Egypt, bringing it under the control of the Roman Restriction Endonucleases: Molecular Scissors for Specifically Cutting DNA. The Science Creative Quarterly. Retrieved on 2008-06-06. 2008 ( MMVIII) is the current year in accordance with the Gregorian calendar, a Leap year that started on Tuesday of the Common Events 1508 - Maximilian I Holy Roman Emperor, is defeated in Friulia by Venetian forces; he is forced to sign a three-year

Databases:

• Roberts RJ, Vincze T, Posfai, J, Macelis D. REBASE. Retrieved on 2008-06-06. 2008 ( MMVIII) is the current year in accordance with the Gregorian calendar, a Leap year that started on Tuesday of the Common Events 1508 - Maximilian I Holy Roman Emperor, is defeated in Friulia by Venetian forces; he is forced to sign a three-year  “Restriction Enzyme Database”

Software:

• Bikandi J, San Millán R, Rementeria A, and Garaizar J. Restriction enzyme digest of DNA. insilico. ehu. es. Retrieved on 2008-06-06. 2008 ( MMVIII) is the current year in accordance with the Gregorian calendar, a Leap year that started on Tuesday of the Common Events 1508 - Maximilian I Holy Roman Emperor, is defeated in Friulia by Venetian forces; he is forced to sign a three-year
• Palmer M. WatCut. University of Waterloo, Ontario, Canada. Retrieved on 2008-06-06. 2008 ( MMVIII) is the current year in accordance with the Gregorian calendar, a Leap year that started on Tuesday of the Common Events 1508 - Maximilian I Holy Roman Emperor, is defeated in Friulia by Venetian forces; he is forced to sign a three-year  “An on-line tool for restriction analysis, silent mutation scanning, SNP-RFLP analysis”
• Vincze,T, Posfai J, Roberts RJ. NEBcutter V2.0. New England Biolabs Inc. . Retrieved on 2008-06-06. 2008 ( MMVIII) is the current year in accordance with the Gregorian calendar, a Leap year that started on Tuesday of the Common Events 1508 - Maximilian I Holy Roman Emperor, is defeated in Friulia by Venetian forces; he is forced to sign a three-year  “Restriction enzyme finder”
• Restriction enzyme digest of DNA software. BioPHP: PHP for Bioinformatics. Retrieved on 2008-06-06. 2008 ( MMVIII) is the current year in accordance with the Gregorian calendar, a Leap year that started on Tuesday of the Common Events 1508 - Maximilian I Holy Roman Emperor, is defeated in Friulia by Venetian forces; he is forced to sign a three-year  “Online tool, free source code”
• pDRAW32. AcaClone software. Retrieved on 2008-06-06. 2008 ( MMVIII) is the current year in accordance with the Gregorian calendar, a Leap year that started on Tuesday of the Common Events 1508 - Maximilian I Holy Roman Emperor, is defeated in Friulia by Venetian forces; he is forced to sign a three-year  “Freeware DNA cloning, sequence analysis and plasmid/DNA plotting software”