RESTRICTION ENZYMES

Introduction

Gene cloning requires that DNA molecules be cut in a very precise fashion. Vector is cut during construction of recombinant DNA molecule. Each vector molecule must be cleaved at a single position, to open up the circle so that new DNA can be inserted. A very special type of nucleases is needed to carry out these manipulations.
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Cont.

Termed as Restriction Restriction Enzymes.

Endonucleases

i.e.

Restriction enzymes are Molecular Scissors that cut DNA into fragments at specific sites in their sequence. These enzymes are the protein produced by bacteria that cleaves DNA at specific sites. Bacteria use restriction enzymes to defend against bacterial viruses called bacteriophages.
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History

Arthur Kornberg and colleagues isolated DNA polymerase (1955). B. Weiss and C.C. Richardson isolated DNA ligase (1966). In 1960's, scientists Stewart Linn , Werner Arber & Dussoix isolated two types of enzymes responsible for phage growth restriction in Escherichia coli (E. coli) bacteria.
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Cont...

They used 32P labeled phages to study the degradation by host bacteria (Restriction).
The first type of enzyme was called a "methylase" while the other was called a "restriction nuclease. H.O. Smith, K.W. Wilcox, and T.J. Kelley isolated and characterized the first sequence specific restriction nuclease (1968) called HindII from Haemophilus influenzae.
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Cont

The 1978 Nobel Prize in Medicine was awarded to Daniel Nathans, Werner Arber and Hamilton Smith for the discovery of restriction endonucleases, leading to the development of recombinant DNA technology. The first practical use of their work was the manipulation of E. coli bacteria to produce human insulin for diabetics.
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Bacteriophage Attack
Infection Destruction of the bacterias DNA

Production of viral parts Lysis

Replication of the viral genome

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Packaging

Repelling Bacteriophage Attack

Methylation sites

Methylase

Repelling Bacteriophage Attack

Methylation sites
Unmethylated methylation sites

Munch! Munch! Munch . . .

Repelling Bacteriophage Attack

Methylation sites

Take that you wicked virus!

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Repelling Bacteriophage Attack

Take that you wicked virus!

Methylase and restriction endonucleases must recognize the same sequences if they are to function as an effective system
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Molecular enzymes used in Gen. Eng.

DNA/RNA polymerases Ligases Topoisomerases Helicases Primases Nucleases

Exonucleases Endonucleases
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Nucleases

Exonucleases, Nucleases that function by removing nucleotides from the ends of the molecule are called exonucleases. Endonucleases, Sequence specific nucleases that break nucleic acid chains somewhere in the interior, rather than at the ends, of the molecule. Term restriction enzyme implies endonucleases in genetic engineering. to
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Nucleases
5 Exonuclease 3 Exonuclease

Endonuclease

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Restriction Endonucleases

Group of enzymes that catalyze the cleavage of DNA at specific sites to produce discrete fragments, used especially in genetic engineering.

Restriction endonucleases are now grouped into different modification systems. Nomenclature system for these enzymes was first given by Smith & Nathans in 1973
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Nomenclature

First letter of the genus & the first 2 letters of the epithet to form a 3 letter abbreviation which identify the species name of the host organism. Strain or type is written as subscript. Roman numericals at the end indicate the chronological sequence of isolation of enzymes from same strain.

Example:

EcoRI

Genus:

Escherichia Species: coli Strain: R First isolated : I

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Cont

When restriction or modification system is derived from virus or plasmid the abbreviated species name of the host is given & extrachromosomal material is identified the subscript. E.g. ECOpi , ECORi

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Types of modification systems

Divided into three types on the basis of, Enzyme complexity Cofactor requirement Position of DNA cleavage Type IMg++, ATP & SAM adenosylmethionine) Type II- Mg++ Type III- Mg++, ATP, stimulation by SAM (S-

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Property
Restriction & Modification

Type I
Single multifunctional enzyme

Type II
Separate nuclease & methylase

Type III
Separate enzymes, Common subunits

Nuclease subunit
Cofactors Cleavage requirements Site of cleavage

Heterodimer
ATP, Mg++, SAM 2 recognition sites in any orientation

Homodimer
Mg++ Single recognition site

Heterodimer
Mg++ (SAM) 2 recognition sites in head to head orientation

Random, At or near 24-26 bps to the 3 approx. 1000 recognition site. side of RS. bp away No Yes Yes

Enzymatic turnover

DNA translocation
Methylation site

Yes
At RS

No
At RS

No
At RS
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Type I

The enzymes are composed subunits, Specificity subunit. Modification subunit Restriction subunit

of

three

In presence of SAM, restriction enzyme binds to its recognition site irrespective of its methylation state
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Cont

Recognition sequence - methylated ATP hydrolysis stimulates dissociation of enzyme from DNA. Recognition site hemi-methylated then ATP stimulates methylation of other strand. SAM acts as methyl donar.

Recognition site cleavage occurs.

unmethylated

DNA

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Type II

The restriction enzyme is independent of its methylase. Cleavage occurs at very specific sites that are within or close to the recognition sequence. The vast majority of known restriction enzymes are of type II, and these enzymes find the most use as laboratory tools. The first to be discovered and utilized was EcoRI.
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Cont

Most type II enzymes cut palindromic DNA sequences

Enzymes from different sources that recognize the same sequence are known as isoschizomers. Isoschizomers need not cut at the same sequence position. e.g. SmaI XmaI CCC C GGG CCGGG
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Mechanism of Action of Type II Enzymes

Type II enzyme have highly conserved catalytic core structurally. Core is composed of five stranded sheet flanked by two -helices
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Cont...

After encountering particular recognition sequence, restriction enzyme undergoes a large conformational change.

It activates the catalytic sites

Enzyme then make one cut on each of the two sugar phosphate backbones.
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Products generated by restriction enzymes

COHESIVE END CUTTERS (staggered cuts): Enzyme Recognition Site Ends of DNA After Cut EcoRI 5GAATTC3 3CTTAAG5 5CTGCAG3 3GACGTC5 5G 3CTTAA 5CTGCA 3G AATTC3 G5 G3 ACGTC5

PstI

BLUNT END CUTTERS (direct cuts): Enzyme Recognition Site Ends of DNA After Cut HaeIII 5GGCC3 3CCGG5 5GG 3CC CC3 GG5
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Some REs and their cleavage patterns

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Restriction enzyme maps

Cleavage of DNA with restriction enzymes provides landmarks and sequence information.

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Frequency of Cutting

Average distance between cuts is, 4n where n is number of bps in recognition site.

e.g. For EcoR1 = 46 = 4096 bps Provided all bases occur equally frequently.

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GC content

These differences between A+T & C+G contents are often expressed as GC content. GC content represent percentage of nucleotides which are either G or C Additionally higher eukaryotic genomes tend to contain fewer CG repeats (Bird 1980). REs will cut DNA relatively infrequently due to this proportion of GC content.
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Type III

Act as complexes of two different subunits, Subunit M, DNA sequence recognition & modification Subunit R, Nuclease action They recognize specific sequence away from recognition sequence (about 24-26bp) to the 3 side of host recognition sequence.

ATP absolute requirement but do not hydrolyse

SAM only stimulates activity; no absolute requirement
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Other Modification Systems

Most E.coli strains used in the lab for DNA isolation contain three site specific DNA methylation system.

Dam methylation Dcm methylation Mcr system

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Cleavage Pattern

Blunt Cut: Staggered Cut:

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Blunt Cut

Many restriction enzymes make a simple double stranded cut in middle of the recognition sequence resulting into blunt ends.

e.g. PvuII, HaeII

HaeII 5.GGCC.3 3.CCGG.5,

5.GGOH.3 3.CCp.5
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Staggered Cut
Dont cut two DNA strands exactly at same nucleotide base pair. Instead the cleavage is usually staggered by 2-4 nucleotides so that the resulting DNA fragments have short single strands over hangs at each end & this is called sticky or cohesive end. As base pairing between them can stick the DNA molecules back together again. The staggered end resulting in over hanging ends either at 5 ( EcoRI) or 3 (PstI). e.g. EcoRI 5.GAATTC.3------------>5.GOH.3 3.CTTAAG.5------------>3.CTTAAp.5. 3 end always carries hydroxyl (OH) group while 5 end always carries phosphate group.
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Recognition Sites

Every restriction enzyme has a specific site of recognition of DNA base pairs. Some recognize the short sequences of 4 8 bp in palindromic pattern e.g. Type II enzymes. Some recognize asymmetric sequences of 5 7 bp e.g. Type I & III enzymes. The enzymes which cleave 4 bp is known as Tetracutter & those which cleaves 6 bp as hexacutters & octacutters for 8 bp cutting enzymes.
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Isoschizomers
It can be define as two enzymes having same recognition site. HindIII .AAGCTT. HsuI .TTCGAA. Some enzymes are there which have same recognition site but different cleavage site. e.g. e.g. SmaI 5.CCCGGG.3 3.GGGCCC.5 5.CCCGGG.3 3.GGGCCC.5

XmaI

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Star Activity

First identified by Polisky et. al. in 1975. Restriction enzymes are heavily influenced by reaction conditions. Deviations from optimal conditions may even alter the cleavage specificity. This is called star activity of the enzyme. Ex.

Eco RI recognizes 5AATT3 instead of 5GAATTC3 when we, Increase the pH Lower concentration of NaCl Replace Mg by Mn in presence of glycerol or methyl sulfoxide. 38

Cont...

The most common types of altered activity are single base substitutions, truncation of the outer bases in the recognition sequence, and single-strand nicking Many other enzymes also show star activity like, AvaI, BamHI, BsuI, EcoRV, PvuII, SaiI, HindIII, HpaI, PstI, TaqI, XbaI etc.

Star activity is also observed under optimum buffer conditions when restriction enzymes are used in high concentration.

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Preventing Star Activity

Star activity is inhibited by, p-chloromercuribenzoate where as the normal activity is not affected.

Also it has shown that, presence of spermine or spermidine in the reaction mixture effectively suppresses the star activity of BamHI, BsuI, EcoRI, EcoRV HindIII, PstI, SaiI.

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Storage of Restriction Enzymes

Critically depends upon following factors, Buffer composition Enzyme concentration Presence of additives Appropriate storage temperature Buffer composition varies with different enzymes. For most enzymes; 10-50mM Tris HCl(pH7.5), minimum 50mMNaCl/KCl, 1mM EDTA & 60% glycerol (v/v) are recommended.
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Cont...

Some enzymes require additives like 4dithiothritol,1,4-dithioerythritol,b-mercaptoethanol. These additives prevent oxidation of cystein residues. Restriction enzymes should be stored in an unfrozen solution at temperature below 00C preferably at -200C in 60% glycerol.
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Thermal Inactivation Of RE

If further manipulations of the digested DNA are to be performed, the RE should be inactivated A simple way to stop restriction reaction is by adding EDTA, which chelates Mg ion, thereby preventing catalysis. Most restriction enzymes can be inactivated by incubation at 650C for 20 minutes.
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Applications of Restriction Enzymes

Site Directed Mutagenesis Polymorphism Studies Library construction and screening Designing & production of gene probes Recombinant DNA technology Restriction Fragment Length Polymorphism

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