Restriction Enzymes

Bacterial restriction enzymes cut the sugar-phosphate backbone of the DNA molecule at specific recognition sequences, so these DNA-cutting enzymes are often called restriction endonucleases. Restriction enzymes hydrolyze the backbone of DNA between deoxyribose and phosphate groups, leaving a phosphate group at the 5' ends and a hydroxyl at the 3' ends of both strands. A few restriction enzymes inefficiently cleave single stranded DNA.

A consensus sequence may be a short sequence of nucleotides that is found several times in the genome and is thought to play the same role in its different locations. Restriction enzymes usually have palindromic consensus recognition sequences, which correspond to the site where they cut the DNA. For example, the bacterium Hemophilus aegypticus produces a restriction endonucleases termed HaeIII that cuts DNA between the adjacent G and C wherever it encounters the sequence:

5'GGCC3'
3'CCGG5'

Some features of recognition sequences:
1. Variable length (4-8 bp). Length of the recognition sequence dictates how frequently the enzyme will cut a random sequence of DNA – on average, every 256 bp for a 4 bp recognition site, every 4096 bp for a 6 bp recognition sequence, and every 48 bp for an 8 bp recognition site.
2. Most sequences are palindromic – displaying the same sequence in both direction
3. Isoschizomers – where different restriction enzymes cut the DNA backbone at different recognition sequences.
4. Ambiguous or unambiguous – ambiguous restriction enzymes cut the DNA at recognition sites that begin and end with specific nucleotid sequences, but which can have variable nucleotides (n, pu, py) inserted between the specific 5' and 3' ends, unambiguous recognition sites have invariable sequences.
5. The recognition site for one restriction endonuclease may include the restriction site for another

Compare:
BamHI GGATCC & CCTAGG
NotI GCGGCCGC & CGCCGGCG
Sau3AI GATC & CTAG
SacI GAGCTC & CTCGAG
SstI GAGCTC & CTCGAG
HinfI GAnTC & CtnAG
XhoII puGATCpy & pyCTAGpu


HaeIII and AluI cut straight across the double helix producing "blunt" ends. However, many restriction enzymes cut in an offset fashion, generating an overlapping segment of single-stranded DNA. Such extensions are called "sticky ends" because they are able to form base pairs with any DNA molecule that contains the complementary sticky end. Such a union can be made permanent by another enzyme, DNA ligase, which forms covalent bonds along the backbone of each strand, producing a molecule of recombinant DNA (rDNA).

AluI and HaeIII produce blunt ends, while BamHI, HindIII, and EcoRI produce offset, “sticky” ends. Restriction enzyme, recognition sequences, bacterial genus and species:

AluI....................................... 5’ … AGcutCT … 3’
Arthrobacter luteus............................. 3’ … TCcutGA … 5’

HaeIII..................................... 5’ … GGcutCC … 3’
Haemophilus influenzae.......................... 3’ … CCcutGG … 5’

BamHI.................................... 5’ … GcutGATCC … 3’
Bacillus amyloliquefaciens................. 3’ … CCTAGcutG … 5’

HindIII.................................... 5’ … AcutAGCTT … 3’
Haemophilus influenzae ....................3’ …TTCGAcutA … 5’

EcoRI..................................... 5’ … GcutAATTC … 3’
Escherichia coli................................... 3’ … CTTAAcutG … 5’


Most restriction enzymes occur in bacteria, where they play a role in defense by chopping up - restricting - the genomes of bacteriophages. In almost all cases, a bacterium that produces a particular restriction endonuclease also synthesizes a companion DNA methyltransferase, which methylates the DNA target sequence for that restriction enzyme, thereby protecting the bacterial genome from cleavage. This combination of restriction endonuclease with methylase is referred to as a restriction-modification system.

The ability to produce recombinant DNA molecules revolutionized the study of genetics, and has proved important to biotechnology. Recombinant DNA technology has been utilized to produce human insulin (for diabetics), human factor VIII (for hemophilia A), and other useful proteins.

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