Restriction Enzymes | Mechanism of Action | With Clear Diagrams

Recombinant DNA is DNA with a new sequence formed by joining fragments from two or more different sources. One of the first
breakthroughs leading to recombinant DNA technology was the
discovery in the late 1960s by Werner Arber and Hamilton Smith
of bacterial enzymes that make cuts in double-stranded DNA.
These enzymes, known as restriction enzymes or restriction endonucleases, recognize and cleave specific sequences about 4 to
8 base pairs long. They normally protect the host cell
by destroying phage DNA after its entrance. Cells protect their
own DNA from restriction enzymes by methylating specific nucleotides. Incoming foreign DNA that is not methylated in the
same pattern as the host may be cleaved by host restriction enzymes. Restriction enzymes recognize specific DNA sequences
called recognition sites. Each restriction enzyme has its own
recognition site. Hundreds of different restriction enzymes have
been purified and are commercially available. Type I
and type III endonucleases identify their unique recognition sites
and then cleave DNA at a defined distance from it. The more common type II endonucleases cut DNA directly at their recognition
sites. These enzymes can be used to prepare DNA fragments containing specific genes or portions of genes. For example, the restriction enzyme EcoRI, isolated by Herbert Boyer in 1969 from
Escherichia coli, cleaves DNA between G and A in the base sequence 5′-GAATTC-3′. Because DNA is antiparallel this sequence is reversed on opposite strands of DNA. When
EcoRI cleaves between the G and A residues, the remaining unpaired 5′-AATTC-3′ remains at the end of each strand. The complementary bases on two EcoRI-cut fragments can hydrogen
bond, thus EcoRI and other endonucleases like it generate cohesive or sticky ends. In contrast, cleavage by restriction enzymes
like AluI and HaeIII leave blunt ends. Note that each
enzyme is named after the bacterium from which it is purified.
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