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Restriction Enzymes: Biochemistry Seminar Fathima I Nazeer February 13 2004

Restriction enzymes are molecular scissors that cut DNA molecules at specific recognition sites. They were discovered in the 1960s by researchers studying bacteria. There are three main types of restriction enzymes that differ in where they cut DNA and how their methylation patterns protect the host DNA. They are important tools in biotechnology and molecular biology as they allow manipulation of DNA fragments through processes like restriction mapping and recombinant DNA techniques.

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42 views17 pages

Restriction Enzymes: Biochemistry Seminar Fathima I Nazeer February 13 2004

Restriction enzymes are molecular scissors that cut DNA molecules at specific recognition sites. They were discovered in the 1960s by researchers studying bacteria. There are three main types of restriction enzymes that differ in where they cut DNA and how their methylation patterns protect the host DNA. They are important tools in biotechnology and molecular biology as they allow manipulation of DNA fragments through processes like restriction mapping and recombinant DNA techniques.

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Ashima Goel
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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RESTRICTION ENZYMES

BIOCHEMISTRY SEMINAR
FATHIMA I NAZEER
February 13th 2004.
What are restriction enzymes?

• Molecular scissors that cut double stranded DNA


molecules at specific points.

• Found naturally in a wide variety of prokaryotes

• An important tool for manipulating DNA.


Discovery

• Arbor and Dussoix in 1962 discovered that


certain bacteria contain Endonucleases which
have the ability to cleave DNA.
• In 1970 Smith and colleagues purified and
characterized the cleavage site of a Restriction
Enzyme.
• Werner Arbor, Hamilton Smith and Daniel
Nathans shared the 1978 Nobel prize for
Medicine and Physiology for their discovery of
Restriction Enzymes.
Biological Role

• Most bacteria use Restriction Enzymes as a


defence against bacteriophages.
• Restriction enzymes prevent the replication of
the phage by cleaving its DNA at specific sites.
• The host DNA is protected by Methylases which
add methyl groups to adenine or cytosine bases
within the recognition site thereby modifying the
site and protecting the DNA.
Types of Restriction Enzymes
Cleavage Location of Examples
methylase
site

Type I Random Endonuclease EcoK I


Around 1000bp and methylase EcoA I
away from located on a
CfrA I
recognition site single protein
molecule
Type II Specific Endonuclease EcoR I
Within the and methylase BamH I
recognition site are separate Hind III
entities

Type III Random Endonuclease EcoP I


and methylase
Recognition sites of most restriction
enzymes have a twofold rotational symmetry

Restriction enzymes have corresponding symmetry to


facilitate recognition and usually cleave the DNA on the axis
of symmetry
Restriction fragments can be blunt ended or
sticky ended

5’ G A A T T C 3’ 5’ G A T A T C 3’
3’ C T T A A G 5’ 3’ C T A T A G 5’

Sticky Ends Blunt Ends

Sticky ends or blunt ends can be used to join DNA


fragments.
Sticky ends are more cohesive compared to blunt ends.
Isoschizomers and Neochischizomers

• Restriction enzymes that have the same recognition sequence as


well as the same cleavage site are Isoschizomers.

• Restriction enzymes that have the same recognition sequence but


cleave the DNA at a different site within that sequence are
Neochizomers. Eg:SmaI and XmaI

CCCGGG CCCGGG
GGGCCC GGGCCC
Xma I Sma I
Mechanism of Action

Restriction Endonuclease scan the length of the


DNA , binds to the DNA molecule when it
recognizes a specific sequence and makes one
cut in each of the sugar phosphate backbones of
the double helix – by hydrolyzing the
phoshphodiester bond. Specifically,the bond
between the 3’ O atom and the P atom is
broken.
Direct hydrolysis by nucleophilic attack at
the phosphorous atom

3’OH and 5’ PO43- is produced. Mg2+ is required for the catalytic


activity of the enzyme. It holds the water molecule in a position
where it can attack the phosphoryl group and also helps polarize the
water molecule towards deprotonation .
Structure of EcoR V endonuclease

• Consists of two subunits


– dimers related by two
fold rotational symmetry.
• Binds to the matching
symmetry of the DNA
molecule at the restriction
site and produces a kink
at the site.
Hydrogen bonding interactions between EcoRv and its DNA
substrate
A comparison of cognate and non-specific DNA in the
EcorV-DNA complex.
Uses of Restriction Enzymes

Restriction Enzymes
can be used to
generate a restriction
map. This can provide
useful information in
characterizing a DNA
molecule.
Uses….
Restriction Fragment Length Polymorphism is a tool to study
variations among individuals & among species
Uses….

Restriction enzymes are


most widely used in
recombinant DNA
technology.
References

• Biochemistry (1995), Wiley & Sons, Inc.


Voet D. and Voet J.G.
• Biochemistry (2002), Freeman & Co.
Berg, J.M., Tymoczco, J.L., Stryer, L.
• An Introduction to Genetic Analysis (2000), Freeman &
Co.
Griffiths, A., Miller, J.H., Suzuki, D.T., Lewontin, R.C., Gelbart, W.M.
• Molecular Cell Biology (2000), Freeman & Co.
Lodish, Berk, Zipursky, Matsudaria, Baltimore, Darnell

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