Enzymes that cut DNA at or near specific recognition nucleotide sequences known as restriction sites. Especial class of enzymes that cleave (cut) DNA at a specific unique internal location along its length. Often called restriction endonucleases (Because they cut within the molecule). Discovered in the late 1970s by Werner Arber, Hamilton Smith, and Daniel Nathans. Essential tools for recombinant DNA technology. Naturally produced by bacteria that use them as a defense mechanism against viral infection. Chop up the viral nucleic acids and protect a bacterial cell by hydrolyzing phage DNA.

1. By
Ananya Azad
Hrisha

2. Overview
• Definition.
• Brief Description.
• Why named so?
• Nomenclature.
• Characteristics.
• Mode of Actions.
• Types.
• Impacts & Uses.
• Use of Restriction Enzymes in Recombinant DNA Technonoly.
• Restriction Enzyme Recognition Sequences.
• Summary.

3. RESTRICTION
ENZYME/RESTRICTION
ENDONUCLEASE
• Enzymes that cut DNA at or near specific recognition nucleotide sequences known as
restriction sites.
• Especial class of enzymes that cleave (cut) DNA at a specific unique internal location
along its length.
• Often called restriction endonucleases (Because they cut within the molecule).
• Discovered in the late 1970s by Werner Arber, Hamilton Smith, and Daniel Nathans.
• Essential tools for recombinant DNA technology.
• Naturally produced by bacteria that use them as a defense mechanism against viral
infection.
• Chop up the viral nucleic acids and protect a bacterial cell by hydrolyzing phage DNA.

4. RESTRICTION
ENZYME
• The bacterial DNA is protected from digestion
because the cell methylates (adds methyl groups to)
some of the cytosines in its DNA.
• The purified forms of these bacterial enzymes are
used in today's laboratories.
• Commonly classified into three types, which differ in
their structure and whether they cut their DNA
substrate at their recognition site, or if the
recognition and cleavage sites are separate from one
another.
• To cut DNA, all restriction enzymes make two
incisions, once through each sugar-phosphate
backbone (i.e. each strand) of the DNA double helix.

6. Nomenclature
• Since their discovery in the 1970s,
many restriction enzymes have
been identified; for example, more
than 3500 different Type II
restriction enzymes have been
characterized.
• Each enzyme is named after the
bacterium from which it was
isolated, using a naming system
based on bacterial genus, species
and strain.
• For example, the name of the
EcoRI restriction enzyme was
derived as shown in the box.
Derivation of the EcoRI name
Abbreviation Meaning Description
E Escherichia genus
co coli specific epithet
R RY13 strain
I First identified
order of identification
in the bacterium

7. Characteristics
• Most restriction enzymes are specific to a
single restriction site
• Restriction sites are recognized no matter
where the DNA came from
• The number of cuts in an organism's DNA made
by a particular restriction enzyme is
determined by the number of restriction sites
specific to that enzyme in that organism's
DNA.
• A fragment of DNA produced by a pair of
adjacent cuts is called a RESTRICTION
FRAGMENT.
• A particular restriction enzyme will typically
cut an organism's DNA in to many pieces, from
several thousand to more than a million!
• There is a great deal of variation in restriction
sites even within a species.
• The enzyme "scans" a DNA molecule,
looking for a particular sequence,
usually of four to six nucleotides.
• Once it finds this recognition
sequence, it stops and cuts the
strands. This is known as enzyme
digestion.
• On double stranded DNA the
recognition sequence is on both
strands, but runs in opposite
directions.
• This allows the enzyme to cut both
strands. Sometimes the cut is blunt,
sometimes the cut is uneven with
dangling nucleotides on one of the
two strands. This uneven cut is
known as sticky ends.
Mode of Action

8. Mode of action (how R.E. cuts
DNA)
• The enzyme makes two incisions, one through each of the sugar-phosphate backbones
(i.e., each strand) of the double helix without damaging the nitrogenous bases.
• Restriction enzymes hydrolyze the backbone of DNA between deoxyribose and
phosphate groups. This leaves a phosphate group on the 5' ends and a hydroxyl on the 3'
ends of both strands. A few restriction enzymes will cleave single stranded DNA, although
usually at low efficiency.
• The restriction enzymes most used in molecular biology labs cut within their
recognition sites and generate one of three different types of ends. In the diagrams
below, the recognition site is boxed in yellow and the cut sites indicated by red triangles.
5' overhangs: The enzyme cuts asymmetrically within the recognition site such that a short
single-stranded segment extends from the 5' ends. BamHI cuts in this manner.

9. Mode of action (how R.E. cuts
DNA)
 Blunts: Enzymes that cut at precisely opposite sites in the two strands of DNA generate
blunt ends without overhangs. SmaI is an example of an enzyme that generates blunt
The 5' or 3' overhangs generated by enzymes that cut asymmetrically are called sticky ends or
cohesive ends, because they will readily stick or anneal with their partner by base pairing. The
sticky end is also called a cohesive end or complementary end in some reference.
3' overhangs: Again, we see asymmetrical cutting within the recognition site, but the
result is a single-stranded overhang from the two 3' ends. KpnI cuts in this manner.

10. Kinds of Restriction
Enzymes

11. Star Activity of Restriction
Enzymes
Star activity is defined as the alteration in the digestion specificity that occurs under sub-optimal
enzyme conditions. Star activity results in cleavage of DNA at non-specific sites. Some of the sub-
optimal conditions that result in star activity are as follows:
• pH >8.0
• glycerol concentration of >5%
• enzyme concentration >100 units/mg of DNA
• increased incubation time with the enzyme
• presence of organic solvents in the reaction mixture
• incorrect cofactor or buffer
Type V
Type V restriction enzymes (e.g., the cas9-gRNA complex from CRISPRs) utilize guide RNAs to target specific non-palindromic
sequences found on invading organisms. They can cut DNA of variable length, provided that a suitable guide RNA is provided.
The flexibility and ease of use of these enzymes make them promising for future genetic engineering applications

12. Artificial restriction
enzymes
 Artificial restriction enzymes can be generated by fusing a natural or
engineered DNA binding domain to a nuclease domain (often the
cleavage domain of the type IIS restriction enzyme FokI).
 Such artificial restriction enzymes can target large DNA sites (up to 36
bp) and can be engineered to bind to desired DNA sequences.
 Zinc finger nucleases - are the most commonly used artificial restriction
enzymes and are generally used in genetic engineering applications, but
can also be used for more standard gene cloning applications.
 Other artificial restriction enzymes are based on the DNA binding
domain of TAL effectors.
 CRISPR RNA molecules are also Artificial restriction enzymes.

13. Restriction Enzyme Recognition
Sequences
• The length of restriction recognition sites varies: The enzymes
EcoRI, SacI and SstI each recognize a 6 base-pair (bp) sequence of
DNA, whereas NotI recognizes a sequence 8 bp in length, and the
recognition site for Sau3AI is only 4 bp in length. Length of the
recognition sequence dictates how frequently the enzyme will
cut in a random sequence of DNA. Enzymes with a 6 bp
recognition site will cut, on average, every 46 or 4096 bp; a 4 bp
recognition site will occur roughly every 256 bp.
• Different restriction enzymes can have the same recognition
site - such enzymes are called isoschizomers: Look at the
recognition sites for SacI and SstI - they are identical. In some
cases isoschizomers cut identically within their recognition site,
but sometimes they do not. Isoschizomers often have different
optimum reaction conditions, stabilities and costs, which may
influence the decision of which to purchase.

15. Restriction Enzyme Recognition
Sequences
• Restriction recognitions sites can be unambiguous or ambiguous: The enzyme BamHI recognizes
the sequence GGATCC and no others - this is what is meant by unambiguous. In contrast, HinfI
recognizes a 5 bp sequence starting with GA, ending in TC, and having any base between (in the
table, "N" stands for any nucleotide) - HinfI has an ambiguous recognition site. XhoII also has an
ambiguous recognition site: Py stands for pyrimidine (T or C) and Pu for purine (A or G), so XhoII
will recognize and cut sequences of AGATCT, AGATCC, GGATCT and GGATCC.
• The recognition site for one enzyme may contain the restriction site for another: For example,
note that a BamHI recognition site contains the recognition site for Sau3AI. Consequently, all
BamHI sites will cut with Sau3AI. Similarly, one of the four possible XhoII sites will also be a
recognition site for BamHI and all four will cut with Sau3AI.
• Other point to notice from the table above is that most recognition sequences are palindromes -
they read the same forward (5' to 3' on the top strand) and backward (5' to 3' on the bottom
strand). Most, but certainly not all recognition sites for commonly-used restriction enzymes are
palindromes. Most restriction enzymes bind to their recognition site as dimers (pairs), as
depicted for the enzyme PvuII in the figure to the right.

16. THE IMPACT OF RESTRICTION ENZYMES
Genetic engineering
• Type II enzymes yielded many practical benefits, as E. coli K12, its genes and its vectors
became the workhorses of molecular biology in the 1970s for cloning, generation of libraries,
DNA sequencing, detection and overproduction of enzymes, hormones, etc.
• The applications of Type II enzymes continued to expand, especially after the arrival of
synthetic DNA, in vitro packaging of DNA in phage particles and improved bacterial hosts and
vectors for overexpression and stabilization of proteins.
• Production of insulin from recombinant bacteria and yeast by Genentech, thus greatly increasing the
supply for diabetics and the production of a recombinant vaccine for Hepatitis B by Biogen to treat the
hundreds of millions of people at risk of infection by this virus.
• Zinc-finger nucleases and the TAL-effector nucleases, which have potential for gene targeting and gene
therapy.

17. THE IMPACT OF RESTRICTION ENZYMES
• Restriction enzymes are tools for monitoring Restriction Fragment Length Polymorphisms
(RFLP), allowing the location of mutations, generation of human linkage maps, identification
of disease genes (such as sickle cell trait or Huntington disease), and last, but not least, the
DNA fingerprinting technique developed by Alec Jeffreys.
DNA fingerprinting
• DNA fingerprinting allows the solution of paternity cases, the identification of criminals and
their victims and the exoneration of the falsely accused. The use of REases in this system
enabled the creation of suitable procedures for such identification.
• Useful for identifying pathogenic bacterial strains, most recently of S. aureus sp with
antibiotic-resistance and virulence factors mediated by mobile genetic elements, e.g. the
methicillin-resistant S. aureus (MRSA) bacteria.

18. Why restriction enzymes are
important for rDNA techniques?
• Restriction enzyme recognizes and cuts, or digests, only one particular
sequence of nucleotide bases in DNA
• Typical restriction enzymes used in cloning experiments recognize four-, six-,
or eight-base sequences.
• It cuts this sequence in the same way each time.
• Hundreds of restriction enzymes are known, each producing DNA fragments
with characteristic ends.

19. The role of a
restriction enzyme in
making recombinant
DNA

20. Conclusion

21. Reference
• https://en.wikipedia.org/wiki/Restriction_enzyme
• Lehninger’s Principles of Biochemistry
• http://www.biology-pages.info/R/RestrictionEnzymes.html
• https://www.neb.com/products/restriction-endonucleases/restriction-endonucleases/types-of-restriction-
endonucleases
• http://www.bio.miami.edu/dana/dox/restrictionenzymes.html
• http://www.bio.miami.edu/dana/dox/restrictionenzymes.html
• https://www.dnalc.org/resources/animations/restriction.html
• http://www.vivo.colostate.edu/hbooks/genetics/biotech/enzymes/renzymes.html
• http://www.biologydiscussion.com/dna/restriction-enzymes/restriction-enzymes-in-dna-mode-of-action-
and-its-types-2/12065
• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3874209/
• http://www.sigmaaldrich.com/technical-documents/articles/biology/restriction-enzymes.html