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    Restriction Enzyme

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    Fawaz Mohamad
    Restriction enzymes are enzymes found in bacteria that cut DNA at specific nucleotide sequences known as restriction sites. They function as a defense mechanism in bacteria by selectively cutting foreign DNA like that from viruses. Over 3000 restriction enzymes have been identified that recognize 4-6 base pair palindromic sequences and make staggered cuts within or near these sites. They are widely used in techniques like restriction fragment length polymorphism and recombinant DNA technology.

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    Restriction Enzyme

    Uploaded by

    Fawaz Mohamad
    375 views33 pages
    Restriction enzymes are enzymes found in bacteria that cut DNA at specific nucleotide sequences known as restriction sites. They function as a defense mechanism in bacteria by selectively cutting foreign DNA like that from viruses. Over 3000 restriction enzymes have been identified that recognize 4-6 base pair palindromic sequences and make staggered cuts within or near these sites. They are widely used in techniques like restriction fragment length polymorphism and recombinant DNA technology.

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    Restriction enzymes are enzymes found in bacteria that cut DNA at specific nucleotide sequences known as restriction sites. They function as a defense mechanism in bacteria by selectively cutting foreign DNA like that from viruses. Over 3000 restriction enzymes have been identified that recognize 4-6 base pair palindromic sequences and make staggered cuts within or near these sites. They are widely used in techniques like restriction fragment length polymorphism and recombinant DNA technology.

    Copyright:

    © All Rights Reserved
    Download as PPT, PDF, TXT or read online from Scribd
    Download as ppt, pdf, or txt
    375 views33 pages

    Restriction Enzyme

    Uploaded by

    Fawaz Mohamad
    Restriction enzymes are enzymes found in bacteria that cut DNA at specific nucleotide sequences known as restriction sites. They function as a defense mechanism in bacteria by selectively cutting foreign DNA like that from viruses. Over 3000 restriction enzymes have been identified that recognize 4-6 base pair palindromic sequences and make staggered cuts within or near these sites. They are widely used in techniques like restriction fragment length polymorphism and recombinant DNA technology.

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    © All Rights Reserved
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    of 33

    Restriction Enzymes

    • What are restriction enzymes?


    Restriction Enzyme
    • Enzyme that cuts DNA at specific nucleotide
    sequences known as restriction sites.
    • Found in bacteria and have evolved to provide
    a defense mechanism against invading viruses.
    • In bacteria they selectively cut up foreign DNA
    in a process called restriction
    • To cut the DNA, restriction enzyme makes two
    incisions, each strand of the DNA double helix.
    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
    site methylase
    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


    24-26 bp away and methylase Hinf III
    from recognition located on a EcoP15 I
    site single protein
    molecule
    Restriction Enzymes
    • Over 3000 have been identified
    • More than 600 available commercially
    • Routinely used for DNA modification and
    manipulation in laboratories.
    • http://en.wikipedia.org/wiki/Restriction_enzyme
    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
    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
    • Restriction Enzymes scan the DNA sequence
    • Find a very specific set of nucleotides
    • Make a specific cut
    Restriction enzymes recognize and
    make a cut within specific
    palindromic sequences, known as
    restriction sites, in the DNA. This is
    usually a 4- or 6 base pair sequence.
    Each of the double
    strands of the DNA
    molecule is
    complimentary to the
    other; thus adenine
    pairs with thymine,
    and guanine with
    cytosine.
    Hae III
    HaeIII is a restriction enzyme that
    searches the DNA molecule until it finds
    this sequence of four nitrogen bases.

    5’ TGACGGGTTCGAGGCCAG 3’
    3’ ACTGCCCAAGGTCCGGTC 5’
    Once the recognition site was found
    HaeIII could go to work cutting
    (cleaving) the DNA

    5’ TGACGGGTTCGAGGCCAG 3’
    3’ ACTGCCCAAGGTCCGGTC 5’
    These cuts produce what scientists call
    “blunt ends”

    5’ TGACGGGTTCGAGG CCAG 3’
    3’ ACTGCCCAAGGTCC GGTC 5’
    “blunt ends” and “sticky ends”
    Remember how HaeIII produced a “blunt end”?
    EcoRI, for instance, makes a staggered cut and
    produces a “sticky end”
    5’ GAATTC 3’
    3’ CTTAAG 5’
    5’ GAATTC 3’
    3’ CTTAAG 5’
    5’ G AATTC 3’
    3’ CTTAA G 5’
    blunt end

    sticky end
    Some more examples of restriction sites of
    restriction enzymes with their cut sites:

    HindIII: 5’ AAGCTT 3’
    3’ TTCGAA 5’

    BamHI: 5’ GGATCC 3’
    3’ CCTAGG 5’

    AluI: 5’ AGCT 3’
    3’ TCGA 5’
    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 .
    The names for restriction enzymes come from:

    • the type of bacteria in which the enzyme is found


    • the order in which the restriction enzyme was identified
    and isolated.

    EcoRI for example


    R strain of E.coli bacteria
    I as it is was the first E. coli restriction enzyme to
    be discovered.
    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.
    Restriction enzymes recognize and make a cut
    within specific palindromic sequences,
    known as restriction sites, in the genetic
    code. This is usually a 4- or 6 base pair
    sequence.

    Example?
    Picking a palindrome
    Words that read the same forwards as
    backwards

    Hannah hannaH

    Level leveL

    Madam madaM
    Palindromes in DNA sequences
    Genetic palindromes
    5 3’ are similar to verbal
    ’
    palindromes. A
    palindromic sequence
    in DNA is one in which
    the 5’ to 3’ base pair
    3’ 5
    ’ sequence is identical
    on both strands.
    Separating Restriction Fragments, I
    Separating Restriction Fragments, II
    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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