Restriction Endonucleases, Polymerases and Ligases

1. ENZYMES IN GENETIC
ENGINEERING
Dr. ESTHER SHOBA R
ASSISTANT PROFESSOR
DEPARTMENT OF LIFE SCIENCES
KRISTU JAYANTI COLLEGE

2. SUB TOPICS
• Nucleases: Exonucleases and Endonucleases
• Polymerases
• DNA ligase

3. Nucleases: Exonucleases and Endonucleases
• The ability to manipulate DNA in vitro depends entirely on the availability of purified enzymes
that can cleave, modify and join the DNA molecule in specific ways.
• At present, no chemical method can achieve the ability to manipulate the DNA in vitro in a
predictable way.
• Only enzymes are able to carry out the function of manipulating the DNA. Each enzyme has a
vital role to play in the process of genetic engineering.
• The various enzymes used in genetic engineering are as follows:
• Nucleases
• Restriction enzymes
• DNA modifying enzymes
• DNA Ligase
• Polymerase
• Topoisomerase

4. NUCLEASES
• ‘Nucleases degrade DNA molecules by breaking the phosphodiester bonds that link one nucleotide to the next in a DNA
strand.
• In addition to their important biological role, nucleases have emerged as useful tools in laboratory studies, and have led
to the development of such fields as recombinant DNA technology, molecular cloning, and genomics’.
• “Processes under control of nucleases are for example
• protective mechanisms against "foreign" (invading) DNA,
• degradation of host cell DNA after virus infections,
• DNA repair,
• DNA recombination,
• DNA synthesis
• DNA packaging in chromosomes and viral compartments,
• maturation of RNAs or RNA splicing.

5. NUCLEASES - CLASSIFICATION
• They are classified by their specificity of their requirement for either a free end (exo)
to start working or they start from anywhere within a molecule (endo) even when no
free ends are available as for example in a covalently closed circle
• EXONUCLEASES:
• Exonucleases catalyses hydrolysis of terminal nucleotides from the end of DNA or RNA
molecule either 5’to 3’ direction or 3’ to 5’ direction. Example: exonuclease I,
exonuclease II etc.
• The main distinction between different exonucleases lies in the number of strands
that are degraded when a double-stranded molecule is attacked.
• For example B al31 degrades both strand
• E. coli exonuclease III degrades only one strand and only from the 3′ terminus.

6. Figure 4.1 The reactions catalysed by the two different kinds of
nuclease. (a) An exonuclease, which removes nucleotides from
the end of a DNA molecule. (b) An endonuclease, which breaks
internal phosphodiester bonds.

9. ENDONUCLEASES
• Endonucleases can recognize specific base sequence (restriction site)
within DNA or RNA molecule and cleave internal phosphodiester bonds
within a DNA molecule.
• Example: E coRI, H ind III, B amHI etc.

10. RESTRICTION ENZYMES
• DNases which act on specific positions or sequences on the DNA are called as
restriction endonucleases.
• The sequences which are recognized by the restriction endonucleases or restriction
enzymes (RE) are called as recognition sequences or restriction sites. These sequences
are palindromic sequences.
• The discovery of these enzymes, led to Nobel Prizes for W. Arber, H. Smith, and D.
Nathans in 1978
• Restriction endonucleases are synthesized by many, perhaps all, species of bacteria:
over 2500 different ones have been isolated and more than 300 are available for use
in the laboratory.

12. MODE OF ACTION
• The restriction enzyme binds to the recognition site and checks for the
methylation (presence of methyl group on the DNA at a specific
nucleotide). If there is methylation in the recognition sequence, then, it
just falls off the DNA and does not cut.
• If only one strand in the DNA molecule is methylated in the recognition
sequence and the other strand is not methylated, then RE (only type I and
type III) will methylate the other strand at the required position.
• The methyl group is taken by the RE from S-adenosyl methionine by using
modification site present in the restriction enzymes.

13. MODE OF ACTION
• However, type II restriction enzymes take the help of another enzyme called
methylase, and methylate the DNA.
• Then RE clears the DNA. If there is no methylation on both the strands of DNA,
then RE cleaves the DNA.
• It is only by this methylation mechanism that, RE, although present in bacteria,
does not cleave the bacterial DNA but cleaves the foreign DNA.
•
• But there are some restriction enzymes which function exactly in reverse mode.
• They cut the DNA if it is a methylate.

14. NOMENCLATURE OF RESTRICTION ENZYMES
• As a large number of restriction enzymes have been discovered, a
uniform nomenclature system is adopted to avoid confusion.
• This nomenclature was first proposed by Smith and Nattens in 1973.
• Every restriction enzyme would have a specific name which would
identify it uniquely.
• The first three letters, in italics, indicate the biological source of the
enzymes, the first letter being the initial of the genus and the second and
third being the first two letters of the species name.

15. NOMENCLATURE OF RESTRICTION ENZYMES
• Thus restriction enzymes from Escherichia coli are called Eco;
• Haemophilus influenzae becomes Hin;
• Diplocococcus pneumoniae Dpn and so on.
• Then comes a letter that identifies the strain of bacteria; Eco R for strain
R.
• Finally there is a roman numeral for the particular enzyme if there are
more than one in the strain in question;
• Eco RI for the first enzyme from E. coli R, Eco RII for the second.

17. RESTRICTION SITES
• Restriction enzymes usually recognize a specific DNA sequence of 4, 5, 6 nucleotides
in length and cleave the DNA within the restriction site.
• There are 4 bases in DNA, randomly distributed.
• The expected frequency of any particular sequence can be calculated as 4 n where n
is the length of recognition sequence.
• Thus tetranucleotide sites will occur every 256 base pair, pentanucleotide sites will
occur every 1025 base pair and hexanucleotide sites will occur every 4096 base pairs.
• The complementary sequences are also known as palindrome sequences or
palindromes.

18. RESTRICTION SITES
• Recognition sites are the palindromes or palindromic sequences.
• Palindromes are the nucleotide-pair sequences that are the same when read forward
(left to right) or backward (right to left) from a central axis of symmetry i.e. two
strands are identical when both are used in the same polarity i.e. in 5’ 3’ direction.
• For example the phrase shown here reads the same in either of the directions (left to
right and right to left):
•
• ANDMADAMDNA
• 5’- GAATTC-3’
• 3’ CTTAAG-5’

19. TYPES OF RESTRICTION ENDONUCLEASES
• The restriction endonucleases can be divided into three groups as type I,
II and III.
• Types I and III have an ATP dependent restriction activity and a
modification activity resident in the same multimeric protein.
• Both these types recognize unmethylated recognition sequences in DNA.
• Type I enzymes cleave the DNA at random site, whereas Type III cleave at
a specific site.
• Type II restriction modification system possess separate enzymes for
endonuclease and methylase activity and are the most widely used for
genetic manipulation.

20. TYPE I RESTRICTION ENDONUCLEASES
• These restriction enzymes recognize the recognition site, but cleave the
DNA somewhere between 400 base pairs (bp) to 10,000 bp or 10 kbp
right or left.
• The cleavage site is not specific.
• These enzymes are made up of three peptides with multiple functions.
• These enzymes require Mg++, ATP and S adenosyl methionine for
cleavage or for enzymatic hydrolysis of DNA.
•
• These enzymes are studied for general interest rather than as useful tools
for genetic engineering.

21. TYPE I RESTRICTION ENDONUCLEASES
• These enzymes are composed of mainly three subunits, a specificity
subunit that determines the DNA recognition site, a restriction subunit,
and a modification subunit
• The recognition site is asymmetrical and is composed of two specific
portions in which one portion contain 3–4 nucleotides while another
portion contain 4–5
• EXAMPLE: EcoK

22. TYPE II RESTRICTION ENZYMES
• Restriction and modification are mediated by separate enzymes so it is
possible to cleave DNA in the absence of modification.
• Although the two enzymes recognize the same target sequence, they can
be purified separately from each other.
• Cleavage of nucleotide sequence occurs at the restriction site.
• These enzymes are used to recognize rotationally symmetrical sequence
which is often referred as palindromic sequence.

23. TYPE II RESTRICTION ENZYMES
• These palindromic binding site may either be interrupted (e.g. BstEII
recognizes the sequence 5´-GGTNACC-3´, where N can be any nucleotide)
or continuous (e.g. KpnI recognizes the sequence 5´-GGTACC-3´).
• They require only Mg2+ as a cofactor and ATP is not needed for their
activity.
• Type II endonucleases are widely used for mapping and reconstructing
DNA in vitro because they recognize specific sites and cleave just at these
sites.

24. STEPS INVOLVED
• These enzymes have nonspecific contact with DNA and initially bind to
DNA as dimmers.
• The target site is then located by a combination of linear diffusion or
“sliding” of the enzyme along the DNA over short distances, and
hopping/jumping over longer distances.
• Once the target restriction site is located, the recognition process
(coupling) triggers large conformational changes of the enzyme and the
DNA, which leads to activation of the catalytic center.
• Catalysis results in hydrolysis of phosphodiester bond and product
release.

25. STEPS INVOLVED

26. BLUNT ENDED FRAGMENTS
• Blunt end cutters Type II restriction enzymes of this class cut the DNA
strands at same points on both the strands of DNA within the
recognition sequence.
• The DNA strands generated are completely base paired. Such
fragments are called as blunt ended or flush ended fragments.

27. STICKY ENDED FRAGMENTS
• Cohesive end cutter Type II restriction enzymes of this class cut the DNA
stands at different points on both the strands of DNA within the recognition
sequence.
• They generate a short single-stranded sequence at the end.
•
• This short single strand sequence is called as sticky or cohesive end.
• This cohesive end may contain 5 -PO 4 or 3 -OH, based upon the terminal
molecule (5 -PO4 or 3 -OH).
• These enzymes are further classified as 5end cutter (if 5 -PO 4 is present) or 3 -
end cutter (if3' -OH is present).

29. Type III RESTRICTION ENZYMES
• The Type III enzyme is made up of two sub-units, one specifies for site
recognition and modification and the other for cleavage.
• In a reaction, it moves along the DNA and requires ATP as source of
energy and Mg++ as co-factor.
• ATPase activity is lacking in these enzymes.
• Some examples of Type III enzyme are HpaI, MboII, FokI, and the like.
They have symmetrical recognition sites and cleave DNA at specific non-
palindromic sequences.
• For cleaving double stranded DNA two sites in opposite orientation must
be present.
• One strand of double stranded DNA is cleaved about 25-27 bp away from
the recognition site which is located in its immediate vicinity.

31. APPLICATIONS OF RESTRICTION ENZYMES
• In various applications related to genetic engineering DNA is cleaved by using these
restriction enzymes.
• They are used in the process of insertion of genes into plasmid vectors during gene
cloning and protein expression experiments.
• Restriction enzymes can also be used to distinguish gene alleles by specifically
recognizing single base changes in DNA known as single nucleotide polymorphisms
(SNPs).
• This is only possible if a mutation alters the restriction site present in the allele.
• Restriction enzymes are used for Restriction Fragment Length Polymorphism (RFLP)
analysis for identifying individuals or strains of a particular species.

32. RESTRICTION ENZYMES
• Different restriction enzymes present in different bacteria can recognize different or
same restriction sites.
• But they will cut at two different points within the restriction site. Such restriction
enzymes are called as isoschizomers.
• Interestingly no two restriction enzymes from a single bacterium will cut at the same
restriction site.

33. DNA LIGASES
• They are also called DNA joining enzymes. DNA ligase forms a specific
type of enzyme in molecular biology, which that facilitates the joining of
the DNA strands together by catalyzing the formation of a
phosphodiester bond.
• Mertz and Davis (1972) for the first time demonstrated that cohesive
termini of cleaved DNA molecules could be covalently sealed with E. Coli
DNA ligase and were able to produce recombinant DNA molecules.
• It plays an important role in repairing single-strand breaks in duplex DNA
in living organisms, but some forms such as DNA ligase IV) may
specifically repair double-strand breaks (i.e. a break in both
complementary strands of DNA)

34. DNA LIGASES
• Single-strand breaks are repaired by DNA ligase using the complementary strand of
the double helix as a template with DNA ligase creating the final phosphodiester bond
to fully repair the DNA.
• DNA ligase has applications in both DNA repair and DNA replication.
• In addition, DNA ligase has extensive use in molecular biology laboratories for
recombinant DNA experiments.
• Purified DNA ligase is used in gene cloning to join DNA molecules together to form
recombinant DNA.

36. MECHANISM OF LIGASE ACTION
• The mechanism of DNA ligase is to form two covalent Phosphodiester bonds between
3` hydroxyl ends of one nucleotide, ("acceptor") with the 5` phosphate end of another
of another ("donor").
• ATP is required for the ligase reaction, which proceeds in three steps:
• 1) adenylation (addition of AMP) of a lysine residue in the active center of the
enzyme, pyrophosphate is released;
• 2) transfer of the AMP to the 5' phosphate of the so-called donor, formation of a
pyrophosphate bond;
• 3) formation of a phosphodiester bond between the 5' phosphate of the donor and
the 3’ hydroxyl of the acceptor.

40. TYPES OF DNA LIGASES

41. E.COLI DNA LIGASE
• The E.coli DNA ligase is encoded by the lig gene.
• DNA ligase in E.coli, as well as most prokaryotes, uses energy gained by
cleaving nicotinamide adenine dinucleotide (NAD) to create the
phosphodiester bond.
• It does not ligate blunt-ended DNA except under conditions of molecular
crowding with Polyethylene glycol, and cannot join RNA to DNA
efficiently.

42. T4 DNA LIGASE
• The DNA ligase from bacteriophage T4 is the ligase most commonly used in laboratory
research.
• It can ligate cohesive or “sticky ends” of DNA, oligonucleotides, as well as RNA and
RNA-DNA hybrids, but not single-stranded nucleic acids.
• It can also ligate blunt – ended DNA with much greater efficiency than E.coli DNA
ligase.
• Unlike E. coli DNA ligase, T4 DNA ligase cannot utilize NAD and it uses ATP as a
cofactor.
• Some engineering has been done to improve the in vitro activity of T4 DNA ligase.