BCH 302 Molecular Genetics and Genetic Engineering 3/cours/BIOS... · Restriction enzymes can be isolated from bacterial cells and used to manipulate DNA fragments, such as those

BCH 302 – Molecular Genetics and Genetic Engineering

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RESTRICTION ENZYMES

(and other)

DNA MANIPULATING/ MODIFYING ENZYMES

Learning objectives

By the end of this lesson students should be able to

o Define and describe the roles of DNA manipulating/modifying enzymes

o Classify restriction enzymes and describe the mechanism of action

o Know the applications of restriction enzymes in genetic engineering

o Know the factors that affect restriction enzyme cleavage of DNA molecule

Introduction

Restriction enzymes (RE) or restriction endonucleases are proteins naturally produced by

bacteria to fight against bacterial viruses that infect bacteria (bacteriophages or phages) or any

foreign DNA whether it came from a virus or from another strain of bacteria. When a phage

infects a bacterium, it inserts its DNA into the bacterial cell so that it might be replicated. REs

cut phage DNA into many fragments (restriction fragments) at specific sites and prevent it from

replicating and consequently eliminate infecting organisms. Thus, they are so named because of

their ability to restrict or limit, the number of strains of bacteriophage that can infect a

bacterium.

Restriction enzymes can be isolated from bacterial cells and used to manipulate DNA

fragments, such as those that contain genes and are therefore very useful tools of recombinant

DNA technology, or genetic engineering.

Each restriction enzyme recognizes a short specific sequence of nucleotide bases (AGCT). These

regions are called recognition sequences also known as palindrome (a nucleotide sequence in

which the 5’ to 3’ sequence of one strand of a segment of DNA is the same as that of its

complementary strand, and are randomly distributed throughout the DNA. Different bacterial

species make restriction enzymes that recognize different nucleotide sequences. Endonucleases

cleave nucleic acid at internal positions, while exonucleases progressively digest from the ends

of the nucleic acid molecules.

When a restriction endonuclease recognizes a sequence, it snips through the DNA molecule by

catalysing the hydrolysis (splitting of a chemical bond by addition of water molecule) of the

bond between adjacent nucleotides. Bacteria prevent their own DNA from being degraded in


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this manner by masking their recognition sequences. Enzymes called methylases add methyl

group (-CH3) to adenine or cytosine bases within the recognition sequence such that the

restriction enzyme will not recognize it. The process of methylation has been shown to be

carried out by DNA sequence-specific methyltransferase enzymes. The restriction enzyme and

its corresponding methylase constitute the restriction-modification system of a bacterial

species. While in plants and animals the primary methylated based is 5-methylcytosine (m5C),

in bacteria the major methylated base is N6-methyladenine (mA) but N4-methylcytosine (mC) is

also found.

5-methylcytosine N6-methyladenine (mA) N4-methylcytosine

Figure1. Structures of the three primary methylated DNA bases in prokaryotes and eukaryotes

Restriction enzymes can used in combination to digest large DNA molecules into smaller

fragments. Because the enzymes always cut at the same site, DNA from a particular molecule

will generate a reproducible set of fragments.

Some advantages of restriction enzymes include:

i. Each has only one restriction activity

ii. Each cuts in a predictable and consistent manner

iii. They require only magnesium ion as a cofactor, no ATP is needed.

Classification of restriction enzymes

There are four classes of restriction enzymes, designated types I, II, II and IV based upon their

molecular structure and need for specific cofactors.


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Type I endonucleases

They are complex, multisubunit, combination of restriction-and-modification enzymes that cut

DNA at random far from their recognition sequences. They have a molecular weight around

300,000 Daltons, are composed of non-identical sub-units, and require Mg2+, ATP (adenosine

triphosphate), and SAM (S-adenosine-methionine) as cofactors for activity. Type I enzymes are

not particularly useful in gene manipulation since their cleavage site in non-specific and they

possess methylase activity. Thus, they have little practical value as they do not produce distinct

restriction fragments or distinct gel-banding patterns.

Type II enzymes

They are a collection of unrelated proteins (frequently differ in amino acid sequence from one

another) of many different kinds that cut DNA at defined positions close to or within their

recognition sequences producing separate restriction fragments and distinct gel-banding

patterns. Indeed, they are the only class used in the laboratory for routine DNA manipulation

and gene cloning. They are much smaller, with molecular weights in the range of 20,000 to

100,000 Daltons. They have identical sub-units and require only Mg2+ as cofactor.

Type III enzymes

This is a large molecule with molecular weight of around 200,000 Daltons, composed of non-

identical sub-units. These enzymes differ from enzymes of the other two classes in that they

require both Mg2+ and ATP but not SAM as cofactors. Type III endonucleases are the scarcest of

the three types. They cleave outside of their recognition sequences and require two such

sequences in opposite orientation within the same DNA molecule to be able to cut; they rarely

give complete digests. They contain both nuclease and methylase activities and their

recognition sites are not symmetrical.

Type IV enzymes

Type IV enzymes recognize modified typically methylated DNA such as the McrBC and Mrr

systems of E. coli

Types I and III are similar in that both restriction and methylase activities are carried out by one

large enzyme complex, unlike the type II system in which the restriction enzyme is independent

of its methylase.

Type II restriction enzyme also differ from the other two types in that they cleave DNA at

specific sites within the recognition site; the others cleave DNA randomly, sometimes hundreds

of bases from the recognition sequence.


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Table1: Examples of restriction enzymes and their target sequences

(http:/www.accessexcellence.org/AE:AEC:re_chart.php)

Enzyme Organism from which derived Target sequence (cut at*) 5’--˃ 3’

Ava I Anabaena variabilis C* C/T C G A/G G

Bam HI Bacillus amyloliquefaciens G* G A T C C

Bgl II Bacillus globigii A* G A T C T

Eco RI Escherichia coli RY 13 G* A A T T C

Eco RII Escherichia coli R245 *C C A/T G G

HaeIII Haemophilus aegyptius G G * C C

Hha I Haemophilus haemolyticus G C G * C

Hind III Haemophilus inflenzae Rd A* A G C T T

Hpa I Haemophilus parainflenzae G T T * A A C

Kpn I Klebsiella pneumoniae G G T A C * C

Mbo I Moraxella bovis *G A T C

Pst I Providencia stuartii C T G C A * G

Sma I Serratia marcescens C C C * G G G

Sst I Streptomyces stanford G A G C T * C

Sal I Streptomyces albus G G * T C G A C

Taq I Thermophilus aquaticus T * C G A

Xma I Xanthamonas malvacearum C * C C G G G

Mechanism of action of Restriction Enzymes

To date, the actual mechanism by which a restriction enzyme cuts the DNA to which it is bound

has not been established. However, it is believed that hydrolysis mediated by metal ion binding

is the model. Some restriction enzymes will bind one magnesium divalent ion whereas some

will bind two or none at all.


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The action of restriction enzyme differs depending on the enzyme that is involved. Generally

the process consists of the following steps: 1)- recognition of the binding site, 2)- binding of the

enzyme dimer to the DNA, 3)- cleavage of the DNA, and 4)- enzyme release.

As with many proteins that interact with DNA, all restriction endonucleases will bind DNA

specifically and, with much less strength, nonspecifically. Even non-specific DNA binding is

capable of inducing a conformational modification in the restriction enzyme dimer that will

result in the protein adjusting to the surface of the DNA strands. These changes are not the

same as those that occur when the dimer binds to the recognition site.

As the dimer slides along the DNA strands, it searches for recognition elements and, when

these are stumbled upon, an interaction between the protein and the DNA occurs in which the

non-specific complex is converted into specific complex. This requires significant

conformational changes in the protein and the DNA as well as removal of water molecules from

the protein/DNA interface. An intimate contact is then established that is held by 15—20

hydrogen bonds that form the protein and the DNA bases in the recognition site. These are

mediated through specific amino acids, primarily ASP and GLU, held in a proper three-

dimensional configuration. However, differences exist among restriction enzymes depending on

the amount of water that is removed but, in all cases, it is a substantially greater amount than is

expelled during non-specific binding.

Factors that influence restriction enzyme activity

The digestion of DNA with restriction enzymes might not be successful always. Sometimes the

DNA may not be digest at all and or may digest only partially. If the sequence is known,

restriction sites can be predicted with accuracy, but in the laboratory an enzyme may cut more

often than it should or even at the wrong sites. This may not be related to technique but

because the sequence used may be incorrect, or a due to erroneous restriction. Some

commonly encountered factors that influence the action of restriction enzymes are as follows

1) Nature of the DNA (substrate)

The nature of the substrate strongly influences the activity of restriction enzymes. The most

important parameters are:

o Base distribution in natural DNA

o Tertiary structure of DNA

o Base composition of the flanking sequence

o Position of the cleavage site with respect to each other.


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If the DNA has contaminants like phenol, chloroform, alcohol, detergent, EDTA the restriction

enzyme activity will be inhibited.

The efficiency with which a restriction enzyme cuts its recognition sequence at different

locations in piece of DNA can vary 10 to 50-fold. This is apparently due to influences of

sequences bordering the recognition site, which perhaps can either enhance or inhibit enzyme

binding or activity.

A related situation is seen when restriction recognition sites are located at or very close to the

ends of linear fragments of DNA. Most enzymes require a few bases on either side of their

recognition site in order to bind and cleave. For many commercial enzymes has “end

requirements” (number and types of bases that flank the ends of DNA).

2) Buffer composition

Different restriction enzymes have differing preferences for ionic strength (salt concentration)

and major cation (sodium or potassium). A series of 2 to 4 different buffers can handle a large

number of available enzymes but a few require specific buffer environment. Thus, use of the

wrong buffer can lead to poor cleavage rates.

3) Incubation temperature

Most restriction enzyme cut best at 37°C, but there are many exceptions. Enzymes isolated

from thermophilic bacteria cut best at temperature ranging from 50 to 65°C. Some other

enzymes have a very short half-life at 37°C and it is recommended that they be incubated at

25oC.

4) Influence of DNA methylation

Almost all strains of E. coli bacteria used for propagating cloned DNA contain two site specific

DNA methylases:

o Dam methylase adds a methyl group to the adenine in the sequence GATC, yielding a

sequence symbolized as GmATC.

o Dcm methylase methylates the internal cytosine in CC(A/T)GG, generating the sequence

CmC(A/T)GG

The practical importance of this phenomenon is that a number of restriction endonucleases

will not cleave methylated DNA. Thus, if DNA unpredictably does not cut or cuts only partially,

it is important to check that the enzyme in question is not methylation-sensitive.


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5) Star activity

When DNA is digested with certain restriction enzyme under non-standard conditions, cleavage

can occur at sites that differ from the normal recognition sequence – this activity is called “star

activity”. An example of an enzyme that can exhibit star activity is EcoRI, where cleavage can

occur within a number of sequences that differ from the established GAATTC by single base

substitutions.

Non-standard conditions that may induce star activity include:

o High pH (> 8.0) or low ionic strength (e.g. if buffer is not added)

o Glycerol concentrations >5% (enzymes are usually sold as concentrates in 50% glycerol)

o Extremely high concentration of enzyme (>100 U/µg of DNA)

o Presence of organic solvents in the reaction (e.g. ethanol, DMSO)

Digestion with multiple enzymes

Digesting DNA with two enzymes is a routine task, and most often the two enzymes have

different buffer requirements. There are at least three ways to handle this situation:

o Digest with both enzymes in the same buffer. In many cases, even though a given buffer

is not optimal for enzyme, one can still obtain fairly good cleavage rates. Always consult

enzyme manufacturer reference table for the best single buffer for conducting specific

double digests.

o Cut with one enzyme, then alter the buffer composition and cut with the second

enzyme. This usually applies to situations where one enzyme like a low salt buffer and

the other a high salt buffer, in which case you can digest with the first enzyme for a

time, add a calculated amount of concentrated NaCl and cut with the second enzyme.

o Change buffers between digestions with two enzymes. In some cases, two enzymes will

have totally incompatible buffers. In that case, perform one digestion, recover the DNA

(usually by precipitation) and re-suspend in the buffer appropriated for the second

enzyme.

References

Much information was adapted from Devor EJ (1992) Introduction: A brief history of the RFLP.

In: Devor EJ ‘ed.) Molecular Applications in Biological Anthropology. Cambridge: Cambridge

university press, 1-18.


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Other DNA manipulating and DNA modifying enzymes

In genetic engineering, once a pure sample of DNA has been prepared, the next step for a

molecular biologist is to construct the recombinant DNA molecule. This will require a

competent vector, as well as the DNA that has to be cloned/manipulated, which must be

cleaved at specific points and then joined together in a controlled manner.

The cutting and joining DNA molecule are “two well-known” example of DNA manipulating

techniques. Besides cutting and joining, DNA molecule can be shortened, lengthened, copied

into RNA or into new DNA molecule, and interestingly it can also be modified by addition or

removal of specific chemical groups.

These manipulations, all of which can be carried out in the test tube provide the foundation of

not only for gene cloning, but also the studies into DNA biochemistry, gene structure and the

control of gene expression. Almost all DNA manipulative techniques make use of purified

enzymes. Originally, these enzymes exist within the cells and participate in essential cellular

processes such as DNA replication, transcription, breakdown of unwanted or foreign DNA,

repair of mutated DNA and recombination between different DNA molecules. When these

enzymes are isolated and purified from cell extracts under artificial conditions, they are still

capable of performing their natural reactions (or something closely related to them).

DNA manipulating enzymes

DNA manipulative enzymes can be grouped into 5 broad classes depending on the type of

reaction they catalyze:

o Nucleases: enzymes that cut shorten or degrade nucleic acid molecules

o Ligases: enzymes that join nucleic acid molecules together through phosphodiester

bonds

o Polymerases: enzymes that make copies of DNA molecules

o Modifying enzymes: enzymes that remove or add chemical groups to DNA

o Topoisomerases: enzymes that introduce or remove supercoils from covalently closed

circular DNA.

Nucleases

Degrade DNA molecule by breaking the phosphodiester bonds that link one nucleotide to the

next in a DNA strand. Two kinds exist, exonucleases and endonucleases.


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- The exonucleases act by removing nucleotides progressively (one at a time) from the

end of a DNA molecule (e.g. Nuclease BAL31, E.coliExonuclease III etc)

- Endonucleases: Break internal phosphodiester bonds within a DNA molecule (e.g. S1

Nuclease, Mung Bean Nuclease, DNase I, RNase A, Restriction enzyme etc)

RNAses

1. RNAse A: Endoribonuclease that specifically attacks SS RNA and not DNA

2. RNAse H: Endonuclease that digest the RNA of an RNA˜DNA hybrid

Ligases:

Repair single stranded (ss)-break in one of the strands of a double stranded (ds) DNA

molecule and also join together individual DNA molecule or the two cohesive ends of

the same molecule. They act by catalyzing the formation of phosphodiester bonds

between adjacent 3’-OH and 5’-P termini in DNA. In the process, the ligation of

complementary sticky ends is much more efficient compared to the ligation of two blunt

ends because compatible sticky ends can base pair with one another by hydrogen

bonding – forming a relatively stable structure for the enzyme to work on.

Polymerases:

Enzymes that synthesize new strand of DNA complementary to and existing DNA or RNA

template. There are 4 types of DNA Polymerases are used routinely in molecular biology

techniques:

1. DNA Polymerase I (usually from. E. coli & T4 phage: Have 5’3’ exonuclease and

3’5’exonuclease (Enzyme has dual functions – DNA Polymerization and DNA

degradation). DNA polymerase I is commonly used in Nick Translation & Probe

preparation, Repairing of DNA fragments, producing a blunt end DNA from a sticky

ends DNA

2. Klenow fragment DNA Polymerase: Have 5’--> 3’ Polymerase & 3’5’ exonuclease

activities but lacks the 5’3’ exonuclease activity. It can only synthesize a

complementary DNA strand on a single stranded template and is commonly used in

Sanger dideoxy sequencing procedures to synthetize second strand cDNA in cDNA

cloning, filling in the 3’ recessed termini created by digestion of DNA with restriction

enzyme and labelling the termini of DNA fragment (end filing reaction).


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3. Reverse Transcriptase (RNA-dependent DNA polymerase): This enzyme requires RNA

as a template. It has 5’-->3’ polymerase, 5’-->3’ riboexonuclease and 3’-->5’

exoribonuclease activities and commonly used in the synthesis of cDNA for cloning and

labelling the termini of DNA fragment with producing 5’ ends (filling reaction).

4.Taq DNA Polymerase (PCR enzyme): This enzyme has 5’-->3’polymerase activity ONLY,

no 3’-->5’exonuclease (No proofreading activity). It is widely used in PCR reaction

(requiring specific primers). However, the latest version of Taq has Proofreading

activities with even higher polymerase capabilities.

DNA modifying enzymes

These are enzyme that can modify DNA molecules by addition or removal of specific

chemical groups. The most important for this course are.

1. Alkaline phosphatase (AP): Removes the Phosphate group present at the 5’ terminus

of a DNA molecule and thus prevent recircularization of plasmid during cloning work.

2. Polynucleotide Kinase: Add Phosphate groups on to free 5’ termini (reverse of AP).

3. Terminal deoxynucleotidyl transferase: Adds 1 or more deoxynucleotides on to the 3’

terminus of a DNA molecule (3’ tailing reaction).

4. DNA Methylase (dam & dcm): Transfer of methyl group to internal A or C residues in

the specific sequences to produce methylated duplex DNA.

REFERENCES

1. Pingoud A and Jeltsch A. (2001) Structure and function of endonucleases. Nucleic

Acids Research, 29: 3705 – 3727.

2. Pingoud A. and Jeltsch A. (2001) Structure and function of type II rectriction

endonucleases. Nucleic Acids Res.29:3705-3727.

3. StryerLubert, Biochemistry, $th edition.

4. Devor EJ (1992) Introduction: A brief history of the RFLP. In: Devor EJ (ed.) Molecular

Applications in Biological Anthropology. Cambridge: Cambridge University Press,1-

18.


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15) The electrophoretic mobility of proteins on SDS-PAGE is a powerful tool to estimate the

molecular weight of unknown proteins. Discuss the relevance of this statement.

16) Give two separation methods that can be used to estimate the molecular weight of

macromolecules. State the principle of each technique. Compare and contrast the similarities

and differences of these methods.

17) What makes gel filtration chromatography different from ordinary partitioning and

adsorption chromatographic separation procedures? Suppose you used 8.5 g of sephadex G200

in the column, what would be the bed volume?

18) In cellular processes, biological molecules are known to be very specific in the way they

interact with one another to maintain life. Briefly describe in point form, a single step

procedure which you would exploit to purify RNA from a homogenized mixture of cellular

components. State the principle of the method

19) Explain how you would determine the molecular weight of a native polypeptide by

electrophoresis.

20) Describe how you would determine the amount of biomolecules separated based on gel

filtration chromatography and gas chromatography.

21) You have just separated and purified a complex mixture of biomolecules thought to contain

proteins and the presence of the separated components

22) distinguish between western blotting, Northern blotting and Southern blotting. Give the

principle and application of each.

23) Certain drugs such as morphine bind to specific receptors in neural tissue. Design an

experiment to partially purify these receptors; clearly stating the principle of the method of

choice.


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24) One major difficulty in absorption chromatography is the desorption of bound molecules in

the elution column. Explain the different ways of desorbing molecules in different adsorption

chromatography procedures.

Documents

BCH 302 Molecular Genetics and Genetic Engineering 3/cours/BIOS... · Restriction enzymes can be isolated from bacterial cells and used to manipulate DNA fragments, such as those