UNIT – 2Enzymes used in

Genetic Engineering

• Cutting and pasting are two of the first skills children learn, and the tools they use are scissors and glue.

• Similarly, cutting DNA and pasting DNA fragments together typically are among the first techniques learned in the molecular biology lab and are fundamental to all recombinant DNA work.

Such manipulations of DNA are conducted by a toolkit of enzymes:

restriction endonucleases are used as molecular scissors,

DNA ligase functions to bond pieces of DNA together, and

a variety of additional enzymes that modify DNA are used to facilitate the process.

DNA modifying enzymes• Restriction enzymes and DNA ligases represent

the cutting and joining functions in DNA manipulation.

• All other enzymes involved in genetic engineering fall under the broad category of enzymes known as DNA modifying enzymes.

• These enzymes are involved in the degradation, synthesis and alteration of the nucleic acids.

Types of Modifying Enzymes

Nucleases• Nuclease enzymes degrade nucleic acids by

breaking the phosphodiester bond that holds the nucleotides together.

• Restriction enzymes are good examples of endonucleases, which cut within a DNA strand.

• A second group of nucleases, which degrade DNA from the termini of the molecule, are known as exonucleases.

• Apart from restriction enzymes, there are four useful nucleases that are often used in genetic engineering.

• These are – Bal 31 and – Exonuclease III (exonucleases), and – Deoxyribonuclease I (DNase I) and – S1-nuclease (endonucleases).

• These enzymes differ in their precise mode of action and provide the genetic engineer with a variety of strategies for attacking DNA.

Mode of action

(a) Nuclease Bal 31 is a complex enzyme. Its primary activity is a fast-acting 3’ exonuclease, which is coupled with a slow-acting endonuclease. When Bal 31 is present at a high concentration these activities effectively shorten DNA molecules from both termini.

(b) Exonuclease III is a 3’ exonuclease that generates molecules with protruding 5’ termini.

(c) DNase I cuts either single-stranded or double-stranded DNA at essentially random sites.

(d) Nuclease S1 is specific for single-stranded RNA or DNA.

Mode of action of various nucleases.

(a)Nuclease Bal 31 is a complex enzyme. Its primary activity is a fast-acting 3’

exonuclease, which is coupled with a slow-acting endonuclease. When Bal 31 is present at a high concentration these activities effectively shorten DNA molecules from both termini.

(b) Exonuclease III is a 3’ exonuclease that generates molecules with protruding 5’ termini.

(c) DNase I cuts either single-stranded or double-stranded DNA at essentially random sites.

(d) Nuclease S1 is specific for single-stranded RNA or DNA.

• In addition to DNA-specific nucleases, there are ribonucleases (RNases), which act on RNA.

• These may be required for many of the stages in the preparation and analysis of recombinants and are usually used to get rid of unwanted RNA in the preparation.

• However, as well as being useful, ribonucleases can pose some unwanted problems.

• They are remarkably difficult to inactivate and can be secreted in sweat.

Polymerases• Polymerase enzymes synthesise copies of nucleic acid molecules

and are used in many genetic engineering procedures.

• When describing a polymerase enzyme, the terms ‘DNA-dependent’ or ‘RNA-dependent’ may be used to indicate the type of nucleic acid template that the enzyme uses.

• Thus, a

– DNA-dependent DNA polymerase copies DNA into DNA, – an RNA-dependent DNA polymerase copies RNA into DNA, and – a DNA-dependent RNA polymerase transcribes DNA into RNA.

• These enzymes synthesise nucleic acids by joining together nucleotides whose bases are complementary to the template strand bases.

• The synthesis proceeds in a 5’→3’ direction, as each subsequent nucleotide addition requires a free 3’-OH group for the formation of the phosphodiester bond.

• This requirement also means that a short double-stranded region with an exposed 3’-OH (a primer) is necessary for synthesis to begin.

• Polymerases are the copying enzymes of the cell;

• These enzymes are template-dependent and can be used to copy long stretches of DNA or RNA.

• The enzyme DNA polymerase I has, in addition to its polymerase function, 5’→3’ and 3’→5’ exonuclease activities.

• A major use of this enzyme is in the nick translation procedure for radiolabelling DNA.

• Nick translation (or Head Translation) was developed in 1977 by Rigby and Paul Berg.

• It is a tagging technique in molecular biology in which DNA Polymerase I is used to replace some of the nucleotides of a DNA sequence with their labelled analogues, creating a tagged DNA sequence which can be used as a probe in Fluorescent in situ hybridization or blotting techniques.

• It can also be used for radiolabeling

• The 5’→3’ exonuclease function of DNA polymerase I can be removed by cleaving the enzyme to produce what is known as the Klenow fragment.

• This retains the polymerase and 3’→5’ exonuclease activities.

• The Klenow fragment is used where a single-stranded DNA molecule needs to be copied; because the 5’→3’ exonuclease function is missing, the enzyme cannot degrade the non-template strand of dsDNA during synthesis of the new DNA.

• Therefore, the large or klenow fragment of DNA Polymerase I has DNA Ploymerase & 3’→5’ Exonuclease activities, and is widely used in molecular biology

DNA Polymerase I

• DNA Polymerase I, a template-dependent DNA polymerase, catalyzes 5'→3' synthesis of DNA.

• The enzyme also exhibits 3'→5' exonuclease (proofreading) activity, 5'→3' exonuclease activity.

Klenow Fragment• Klenow Fragment is the large fragment

of DNA polymerase I.

• It exhibits 5'→3' polymerase activity and 3'→5' exonuclease (proofreading) activity, but lacks 5'→3' exonuclease activity of DNA polymerase I.

Synthesis of double-stranded DNA from single-stranded templates:

T4 DNA Polymerase• T4 DNA Polymerase, a template-dependent

DNA polymerase, catalyzes 5'-3' synthesis from primed single-stranded DNA.

• The enzyme has a 3'-5' exonuclease activity, but lacks 5'-3' exonuclease activity.

T4 DNA Polymerase

Highlights:• Stronger 3'-5' exonuclease activity on single-

stranded than on double-stranded DNA and greater (more than 200 times) than DNA polymerase I and Klenow fragment

• Active in restriction enzyme, PCR, RT and T4 DNA Ligase buffers

Applications

• Blunting of DNA ends: fill-in of 5'-overhangs or/and removal of 3'-overhangs

• Synthesis of labelled DNA probes by the replacement reaction

T7 DNA Polymerase• T7 DNA Polymerase, a template dependent

DNA polymerase, catalyzes DNA synthesis in the 5'=>3' direction.

• It is a highly processive DNA polymerase allowing continuous synthesis of long stretches of DNA.

• The enzyme also exhibits a high 3'=>5' exonuclease activity towards single and double-stranded DNA.

• Assays at 37°C require only short incubation times

Highlights:

• Strong 3’=>5’ exonuclease activity, approximately 1000-fold greater than Klenow Fragment.

• Active in restriction enzyme buffers

Terminal Deoxynucleotidyl Transferase

• Terminal Deoxynucleotidyl Transferase (TdT),

• Template-independent DNA polymerase, catalyzes the repetitive addition of deoxyribonucleotides to the 3'-OH of oligodeoxyribonucleotides and single-stranded and double-stranded DNA .

• TdT requires an oligonucleotide of at least three nucleotides to serve as a primer.

Reverse transcriptase

• (RTase) is an RNA-dependent DNA polymerase, and therefore produces a DNA strand from an RNA template.

• It has no associated exonuclease activity.

• The enzyme is used mainly for copying mRNA molecules in the preparation of cDNA (complementary or copy DNA) for cloning, although it will also act on DNA templates.

• Reverse transcriptase is a key enzyme in the generation of cDNA; the enzyme is an RNA-dependent DNA polymerase, which produces a DNA copy of an mRNA molecule.

Enzymes that modify the ends of DNA molecules

• The enzymes alkaline phosphatase, polynucleotide kinase (T4 polynucleotide kinase), and terminal transferase act on the termini of DNA molecules and provide important functions that are used in a variety of ways.

• The phosphatase and kinase enzymes, as their names suggest, are involved in the removal or addition of phosphate groups respectively.

• Bacterial alkaline phosphatase (there is also a similar enzyme, calf intestinal alkaline phosphatase) removes phosphate groups from the 5’ ends of DNA.

• The enzyme is used to prevent unwanted ligation of DNA molecules, which can be a problem in certain cloning procedures.

• Terminal transferase (terminal deoxynucleotidyl transferase) repeatedly adds nucleotides to any available 3 terminus.

• The enzyme is mainly used to add homopolymer tails to DNA molecules prior to the construction of recombinants.

• In many applications it is often necessary to modify the ends of DNA molecules using enzymes such as phosphatases, kinases, and transferases.

DNA ligase – joining DNA molecules

• DNA ligase is an important cellular enzyme, as its function is to repair broken phosphodiester bonds that may occur at random or as a consequence of DNA replication or recombination.

• In genetic engineering it is used to seal discontinuities in the sugar—phosphate chains that arise when recombinant DNA is made by joining DNA molecules from different sources.

• It can therefore be thought of as molecular

glue, which is used to stick pieces of DNA together.

• This function is crucial to the success of many experiments, and DNA ligase is therefore a key enzyme in genetic engineering.

• The enzyme used most often in experiments is T4 DNA ligase, which is purified from E. coli cells infected with bacteriophage T4

• Although the enzyme is most efficient when sealing gaps in fragments that are held together by cohesive ends, it will also join blunt-ended DNA molecules together under appropriate conditions.

• The enzyme works best at 37◦C, but is often used at much lower temperatures (4--15◦C) to prevent thermal denaturation of the short base-paired regions that hold the cohesive ends of DNA molecules together.

• The ability to cut, modify, and join DNA molecules gives the genetic engineer the freedom to create recombinant DNA molecules.

• However, once a recombinant DNA fragment has been generated in vitro, it usually has to be amplified so that enough material is available for subsequent manipulation and analysis.

• Amplification usually requires a biological system, unless the polymerase chain reaction (PCR) is used.

• We must, therefore, examine the types of living systems that can be used for the propagation of recombinant DNA molecules.

• DNA ligase is essentially ‘molecular glue’; with

restriction enzymes, it provides the tools for cutting and joining DNA molecules.

Ligases

• Fast and efficient ligation of DNA and RNA.– T4 DNA Ligase– T4 RNA Ligase

T4 DNA Ligase• T4 DNA Ligase catalyzes the formation of a

phosphodiester bond between 5'-phosphate and 3'-hydroxyl termini in duplex DNA or RNA.

• The enzyme repairs single-strand nicks in duplex DNA, RNA, or DNA/RNA hybrids.

• It also joins DNA fragments with either cohesive or blunt termini, but has no activity on single-stranded nucleic acids.

• The T4 DNA Ligase requires ATP as a cofactor.

T4 RNA Ligase• T4 RNA Ligase catalyzes the ATP-dependent

intra- and intermolecular formation of phosphodiester bonds between 5'-phosphate and 3'-hydroxyl termini of oligonucleotides, single-stranded RNA and DNA.

Conclusion• These are the modifying enzymes represent

the cutting and joining functions in DNA manipulation and genetic engineering.

WHAT IS AN ENZYME?

• Enzymes are proteins and certain class of RNA (ribozymes) which enhance the rate of a thermodynamically feasible reaction and are not permanently altered in the process.

Molecular Scissors

Restriction enzymes are molecular scissors

RESTRICTION ENZYMES

• A restriction enzyme (or restriction endonuclease) is an enzyme that cuts double-stranded or single stranded DNA at specific recognition nucleotide sequences known as restriction sites.

Property of restriction enzymes

• They break the phosphodiester bonds that link adjacent nucleotides in DNA molecules.

HOW RESTRICTION ENZYMES WORKS?

• Restriction enzymes recognize a specific sequence of nucleotides, and produce a double-stranded cut in the DNA, these cuts are of two types:

• BLUNT ENDS.

• STICKY ENDS.

Blunt end

Sticky end

BLUNT ENDS• These blunt ended fragments can be joined to

any other DNA fragment with blunt ends.

• Enzymes useful for certain types of DNA cloning experiments

“STICKY ENDS” ARE USEFUL

DNA fragments with complimentary sticky ends can be combined to create

new molecules which allows the creation and manipulation of DNA

sequences from different sources.

• While recognition sequences vary widely , with lengths between 4 and 8 nucleotides, many of them are palindromic.

PALINDROMES IN DNA SEQUENCES

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 (the 5’

and 3’ ends refers to the chemical structure

of the DNA).

PALINDROME SEQUENCES• The mirror like palindrome in which the same forward

and backwards are on a single strand of DNA strand, as in GTAATG

• The Inverted repeat palindromes is also a sequence that reads the same forward and backwards, but the forward and backward sequences are found in complementary DNA strands (GTATAC being complementary to CATATG)

• Inverted repeat palindromes are more common and have greater biological importance than mirror- like palindromes.

Star effect• Optimum conditions are necessary for the

expected result.

• Under extreme conditions such as elevated pH or low ionic strength, RE are capable of cleaving sequences which are similar but not identical to their recognition sequence.

NOMENCLATURE OF RESTRICTION ENZYME• Each enzyme is named after the bacterium from

which it was isolated using a naming system based on bacterial genus, species and strain.

For e.g EcoRI

Derivation of the EcoRI name

Abbreviation Meaning Description

E Escherichia genus

co coli species

R RY13 strain

I First identified order of identificationin the bacterium

TYPES OF RESTRICTION ENZYMES

• Restriction endonucleases are categorized into three general groups.

• Type I• Type II• Type III

TYPES OF RESTRICTION ENZYMES

Type I Type II Type III

Type IVArtificial

restriction enzymes

continue…..These types are categorization based on: • Their composition.• Enzyme co-factor requirement. • The nature of their target sequence.• Position of their DNA cleavage site relative to

the target sequence.

Type I• Capable of both restriction and modification

activities

• The co factors S-Adenosyl Methionine(AdoMet), ATP, and mg++are required for their full activity

• Contain: two R(restriction) subunits two M(methylation) subunits one S(specifity) subunits• Cleave DNA at random length from recognition

sites

Type II• These are the most commonly available and used

restriction enzymes

• They are composed of only one subunit.

• Their recognition sites are usually undivided and palindromic and 4-8 nucleotides in length,

• They recognize and cleave DNA at the same site.

• They do not use ATP for their activity

• They usually require only mg2+ as a cofactor.

Type III• Type III restriction enzymes cut DNA about 20-30

base pairs after the recognition site.

• These enzymes contain more than one subunit.

• And require AdoMet and ATP cofactors for their roles in DNA methylation and restriction

Type IV• Cleave only normal and modified DNA

(methylated, hydroxymethylated and glucosyl-hydroxymethylated bases).

• Recognition sequences have not been well defined

• Cleavage takes place ~30 bp away from one of the sites

ARTIFICIAL RESTRICTION ENZYMES

• Generated by fusing a natural or engineered DNA binding domain to a nuclease domain

• can target large DNA sites (up to 36 bp)

• can be engineered to bind to desired DNA sequences

Examples of Type II restriction enzymes

EcoRI E = genus Escherichia co = species coliR = strain RY13I= first endonuclease

isolated

BamHI B = genus Bacillus am = species

amyloliquefaciens H = strain H I = first endonuclease

isolated

HindIII H = genus Haemophilus in = species influenzae d = strain Rd III = third endonuclease

isolated

Isoschizomer• Restriction enzymes specific to the

same recognition sequence. For example, SphI (CGTAC/G) and BbuI (CGTAC/G) are isoschizomers of each other.

Neoschizomer• Enzyme that recognizes the same

sequence but cuts it differently is a neoschizomer.

• For example, SmaI(CCC/GGG) and XmaI (C/CCGGG) are neoschizomers of each other.

APPLICATIONSThey are used in

gene cloning and protein expression

experiments

Detection of RFLPs

Restriction

enzymes are most widely used in

recombinant DNA technology.

DNA Mapping

Genotype a DNA sample

by SNP

Genetic Engineering

Vectors

• The second step in molecular cloning is to join the passenger DNA to the DNA of a suitable cloning vehicle.

• These vehicles (or vectors) have the property that they replicate themselves and any attached passenger DNA so that the passenger is amplified and can be eventually isolated.

Plasmids

Relatively Small Double-stranded, closed-circular DNA molecules

that exist apart from the chromosomes of their hosts

Naturally occurring plasmids carry one or more genes

For example, some plasmids carry genes which confer resistance to certain antibiotics. Some plasmids may bear genes that code for the restriction and modification enzymes that were discussed previously

• Some may carry genes that direct the synthesis of enzymes that aid in the production of bacterial poisons or antibiotics.

• The most important property of plasmids is that they bear a special region of DNA called an origin of replication, or more simply an origin.

Desirable properties of plasmids

• It should be small(small plasmids replicate faster and require less energy for replication than large ones.

Finally, small plasmids are easier to purify than large ones because they are less fragile.)

• Its DNA sequence should be known

• It should grow to high copy number in the host cell.

• It should contain a selectable marker that allows cells containing the plasmid to be isolated

• There should be a large number of unique restriction sites

Plasmid purificationThe most common method for purifying plasmid DNA involves three steps:

• First, the bacteria are broken open and then it’s DNA isolated.

• Then the DNA is denatured.• Finally, the DNA is renatured and centrifuged.

Some popular plasmids

• pBR 322: The first really useful plasmid for genetic engineering.

• The "B" stands for Bolivar and the "R" for Rodriguez, another scientist in Boyer's laboratory).

• It contains an ampicillin resistance gene and a tetracycline resistance gene

• In addition it has a relaxed origin of replication

• pUC Plasmid:

• About 2.7 kilobase pairs.• These pUC (pronounced PUCK) plasmids• Carry an ampicillin resistance gene and

an origin of replication, both from pBR322

• They also bear a multiple cloning site -- a sequence of DNA that carries many restriction sites (13, in the case of pUC18)

• Multiple cloning site of the pUC plasmids is special because it also codes for a small peptide. This peptide will correct a specific mutation in the chromosomal gene that codes for the enzyme beta-galactosidase.

• Cells that harbor an active beta-galactosidase enzyme can be made to turn blue in the presence of certain substrates.

Bacteriophages

Bacteriophages• Viruses that infect bacterial cells by injecting

their genetic material into the bacterial cell

• Lysis and lysogeny

Reasons why bacteriophage lambda is a good cloning vehicle

• It can accept very large pieces of foreign DNA. About 20kb of DNA

• It has been extensively reworked over the years

Why lambda?• Large pieces of DNA (up to about 20 kilobase

pairs) can be easily cloned in bacteriophage lambda substitution vectors.

• Plasmid vectors are less useful for cloning big passengers.

• But why clone large pieces of DNA in the first place?

• One obvious reason is that some genes are very big and it is advantageous to have them all in one piece

• Another reason for cloning in lambda is the efficiency it offers in DNA transformation.

. DNA cloning using phages as vectors

MODE OF REPLICATION IN PHAGE M13 VECTOR

Cosmid vector

Combine parts of the lambda with parts of plasmids. an origin of replication (ori). a cos site(a sequence yield cohesive end) . an ampicillin resistance gene (amp), restriction sites for cloning Cosmids can carry up to 50 kb of inserted DNA.

Cloning by using Cosmid vectors

APPLICATION• A particular gene can be isolated and its

nucleotide sequence determined• Control sequences of DNA can be

identified & analyzed• Protein/enzyme/RNA function can be

investigated• Mutations can be identified, e.g. gene

defects related to specific diseases• Organisms can be ‘engineered’ for

specific purposes, e.g. insulin production.