Restriction Enzyme.ppt

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 site

Location of methylase

Examples

Type I

Random Around 1000bp away from recognition site

Endonuclease and methylase located on a single protein molecule

EcoK I EcoA I CfrA I

Type II

Specific Within the recognition site

Endonuclease and methylase are separate entities

EcoR I BamH I Hind III

Type III

Random 24-26 bp away from recognition site

Endonuclease and methylase located on a single protein molecule

EcoP I Hinf III EcoP15 I

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 GGGCCC Xma I

CCCGGG GGGCCC 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 3’ ACTGCCCAAGGTCC

CCAG 3’ 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 5 ’

3’

3’

5 ’

Genetic palindromes are similar to verbal palindromes. A palindromic sequence in DNA is one in which the 5’ to 3’ base pair 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