This document discusses different types of restriction enzymes: Type I enzymes cut DNA randomly far from recognition sites and have restriction and methylation activities. Type II enzymes are commonly used and cut at fixed positions near recognition sites. Type III enzymes recognize two non-palindromic sequences and cut downstream. Type IV enzymes recognize methylated DNA. Type V enzymes can cut variable lengths of DNA guided by RNA. Restriction enzymes have various applications including cloning, genotyping, and restriction fragment length polymorphism analysis.

1. RESTRICTION ENZYMES
Dr. ESTHER SHOBA R
Assistant Professor
Department of Life Sciences
Kristu Jayanti College

2. Type I
• • Type I enzymes are complex, multisubunit, combination
restriction-and-modification enzymes that cut DNA at random far
from their recognition sequences.
• • Cleavage occurs a considerable distance from the recognition
sites, rarely less than 400 bp away and up to 7000 bp away.
• • They require both ATP and S-adenosyl-L-methionine to function;
multifunctional protein with both restriction and methylase activities.
• • Type I enzymes are of considerable biochemical interest, but
they have little practical value since they do not produce discrete
restriction fragments.
• Ex: EcoB, EcoK

3. • Type I restriction enzymes possess three
subunits called HsdR, HsdM, and HsdS;
• HsdR is required for restriction digestion;
• HsdM is necessary for adding methyl groups
to host DNA (methyltransferase activity), and
• HsdS is important for specificity of the
recognition (DNA-binding) site in addition to
both restriction digestion (DNA cleavage) and
modification (DNA methyltransferase) activity

4. TYPE II
• • Type II restriction enzymes are the familiar ones used for everyday molecular
biology applications such as gene cloning and DNA fragmentation and analysis
•
• • These enzymes cleave DNA at fixed positions with respect to their recognition
sequence, creating reproducible fragments and distinct gel electrophoresis patterns.
• • These enzymes are used to recognize rotationally symmetrical sequence which
is often referred as palindromic sequence.
• • 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.
• • Over 3,500 Type II enzymes have been discovered and characterized,
recognizing some 350 different DNA sequences.
• Ex: Eco RI, Hind III, Bam HI

7. TYPE III
• • Type III restriction enzymes recognize two separate non-
palindromic sequences that are inversely oriented.
• • They cut DNA about 20–30 base pairs after the recognition site.
• • The enzyme is a hetero oligomeric, multifunctional protein
composed of two subunits Res (P08764) and Mod (P08763).
• • These enzymes require AdoMet and ATP cofactors for their
roles in DNA methylation and restriction digestion, respectively.
• • The Mod subunit recognises the DNA sequence specific for the
system and is a modification methyltransferase; as such, it is
functionally equivalent to the M and S subunits of type I restriction
endonuclease. Res is required for restriction digestion, although it
has no enzymatic activity on its own.
• • Type III enzymes recognise short 5–6 bp-long asymmetric DNA
sequences and cleave 25–27 bp downstream to leave short, single-
stranded 5' protrusions.
• Example: EcoP15

8. Type IV enzymes
• • Type IV enzymes recognize modified,
typically methylated DNA (methylated,
hydroxymethylated and glucosyl-
hydroxymethylated DNA) and are exemplified
by the McrBC and Mrr systems of E. coli.
Example: REase

9. Type V restriction enzymes
• • 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.

10. Property Type I RE Type II RE Type III RE
Abundance Less common than
Type II
Most common Rare
Recognition site Cut both strands at a Cut both strands at
a specific, usually
palindromic recognition
site (4-8 bp)
Cleavage of one
non- specific strand, only 24-26
location > 1000 bp bp downstream of
away from the 3´ recognition
recognition site site
Restriction and Single Separate nuclease Separate enzymes
modification multifunctional and methylase sharing a
enzyme common subunit
Nuclease subunit Heterotrimer Homodimer Heterodimer
structure
Cofactors ATP, Mg2+, SAM Mg2+ Mg2+ (SAM)
DNA cleavage Two recognition Single recognition Two recognition
requirements sites in any site sites in a
orientation head-to-head
orientation

11. Applications of Restriction
Endonucleases
• 1. Restriction enzymes are utilized for gene insertion
into plasmids during cloning and protein expression
experiments.
• 2. Restriction enzymes can also be used to distinguish
gene alleles by specifically recognizing single base
changes in DNA known as single nucleotide
polymorphisms (SNPs). However, this is only possible if
a mutation alters the restriction site of the enzyme.
• 3. REs are used for the Restriction Fragment Length
Polymorphism (RFLP) analysis for identifying strains or
individuals of particular species.

12. STICKY ENDS

13. BLUNT ENDS

15. Ribonucleases (RNases)
• • Ribonucleases (RNases) are a large group of hydrolytic enzymes that
degrade ribonucleic acid (RNA) molecules.
• • These are nucleases that catalyze the breakdown of RNA into smaller
components.
• • These enzymes are present in all living cells and biological fluids,
including prokaryotes and eukaryotes, and perform many important
functions.
• • The enzyme is commonly used in molecular biology for removal of
RNA within DNA and protein samples.
• • RNases are classified as endoribonucleases and exoribonucleases.
• • Endoribonucleases are forms A, P, H, I, III, T1, T2, U2, V1, Phy M, and V.
• • Exoribonucleases include RNase forms PH, II, R, D, and T.
• • These enzymes cleave various RNA species differently.
• • Common RNase subclasses used in molecular biology are RNase A and
RNase H.
• • It cleaves the phosphodiester bond between the 5'-ribose of a
nucleotide and the phosphate group attached to the 3'-ribose of an
adjacent pyrimidine nucleotide.

16. APPLICATIONS
• Plasmid and genomic DNA preparation
• •Removal of RNA from recombinant protein
preparations
• • Mapping single-base mutations in DNA or
RNA.

17. DNA MODIFYING ENZYMES:
• Are one among the DNA manipulative
enzymes. These are the enzymes that modify
the DNA by adding or removing specific
chemical groups.
• They are of three types:
• 1. Terminal Transferase
• 2. Alkaline Phosphatase
• 3. Polynucleotide kinase

18. Terminal Transferse
• It is the enzyme that converts blunt end of DNA
fragments into sticky end.
• If the restriction enzyme cuts DNA forming blunt
ends, then efficiency of ligation is very low. So the
enzyme terminal transferase converts bunt end into
sticky end.
• Terminal transferase enzyme synthesize short
sequence of complementary nucleotide at free ends
of DNA, so that blunt end is converted into sticky end.
• It is also known as DNA nucleotidylexotransferase
(DNTT).
• It is a specialized DNA polymerase expressed in
immature, pre-B, pre-T lymphoid cells, and acute
lymphoblastic leukemia/lymphoma cells.

19. • Terminal Deoxynucleotidyl Transferase (TdT) catalyzes
the incorporation of single deoxynucleotides into the 3'-
OH terminus of single- or double-stranded DNA, making
it an effective agent in 3' end labeling applications such
as Terminal dUTP nick end labeling (TUNEL).
• Terminal Deoxynucleotidyl Transferase (TdT) requires an
oligodeoxynucleotide of at least three bases as a primer,
but is not template-dependent. The preferred substrate
of this enzyme is a 3'overhang, but it can also add
nucleotides to blunt or recessed 3'-ends. Cobalt is a
necessary cofactor. However, the enzyme catalyzes
reaction upon Mg and Mn administration in vitro

22. Alkaline phosphatase
• Alkaline phosphatase is a glycoprotein with two identical subunits. The cohesive
ends of broken plasmids, instead of joining with foreign DNA, join the cohesive
end of the same DNA molecules and get re-circularized. To overcome this problem
the restricted plasmid is treated with an enzyme, alkaline phosphatase, that
digests the terminal phosphoryl group.
• The restriction fragments of the foreign DNA to be cloned are not treated with
alkaline phosphatase.
• Therefore, the 5′ end of foreign DNA fragment can covalently join to 3′ end of the
plasmid. The recombinant DNA thus obtained has a nick with 3′ and 5′ P hydroxy
ends. Ligase will only join 3′ and 5′ ends of recombinant DNA together if the 5′ end
is phosphorylated.
• Thus, alkaline phosphatase and ligase prevent re-circularization of the vector and
increase the frequency of production of recombinant DNA molecules. The nicks
between two 3′ ends fragment and vector DNA are repaired inside the bacterial
cells during the transformation.

26. Polynucleotide kinase:
• Kinase is the group of enzyme, which adds a free
pyrophosphate (PO4) to a wide variety of substrates like
proteins, DNA and RNA.
•
• It uses ATP as cofactor and adds a phosphate by breaking the
ATP into ADP and pyrophosphate.
• It is widely used in molecular biology and genetic engineering
to add radio-labelled phosphates.
• In RDT experiments mostly T4 polynucleotide kinase is used.
•
• It adds phosphate group from ATP molecule to terminal 5’end
after dephosphorylation by alkaline phosphatase.

28. METHYLASES
• Methyltransferase or methylase catalyzes the transfer of methyl group (-
CH3) to its substrate. The process of transfer of methyl group to its
substrate is called methylation.
• • Methylation is a common phenomenon in DNA and protein structure.
• • Methyltransferase uses a reactive methyl group that is bound to
sulfur in S- adenosyl methionine (SAM) which acts as the methyl donor.
• • Methylation normally occurs on cytosine (C) residue in DNA
sequence. In protein, methylation occurs on nitrogen atom either on N-
terminus or on the side chain of protein.
• • DNA methylation regulates gene or silence gene without changing
DNA
• sequences, as a part of epigenetic regulation.
• • In bacterial system, methylation plays a major role in preventing their
genome from degradation by restriction enzymes. It is a part of restriction –
modification system in bacteria.