Enzymes for Nucleic

Acid Manipulation
Mika Yssa F. Marcelino
Restriction Enzymes
(RE)
Restriction Enzymes
 Restriction enzymes are also called "molecular scissors" as they cleave
DNA at or near specific recognition sequences known as restriction
sites.
 These enzymes make one incision on each of the two strands of DNA
and are also called restriction endonucleases.
 Each restriction enzyme recognizes specific DNA sequences, and
cleavage can occur within the recognition sequence or some distance
away, depending on the enzyme. The recognition sequences are
generally 4 to 8 base pairs (bp) in length, and cleavage can produce
sticky ends (5′ or 3′ protruding ends) or blunt ends
 Most bacteria use restriction enzymes as defense against
bacteriophages.
Types of Restriction Enzymes
1. Type I Restriction Endonucleases
▪ These enzymes have both restriction and modification
activities.
▪ Restriction depends upon the methylation status of the
target DNA.
▪ The cleavage occurs approximately 1000 bp away from the
recognition site.
▪ 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 nucleotides.
 Types of Restriction Enzymes
2. Type II Restriction Endonucleases
▪ 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.
▪ The 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.
▪ These palindromic binding sites may either be interrupted or continuous.
▪ 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.
Types of Restriction Enzymes
3. Type III Restriction Endonucleases
▪ These enzymes recognize and methylate the same DNA
sequence but cleave 24–26 bp away.
▪ They have two different subunits, in which one subunit (M)
is responsible for recognition and modification of DNA
sequence and other subunit (R) has nuclease action.
▪ Mg2 + ions, ATP are needed for DNA cleavage and process
of cleavage is stimulated.
▪ This enzyme make a cleave only in one strand.
▪ Two recognition sites in opposite orientation are necessary
to break the DNA duplex.
NOMENCLATURE
 The naming convention takes into account three
characteristics of the enzyme’s organismal origin—genus,
species, and strain or serotype—to develop a shortened
name followed by roman numerals to represent multiple
restriction enzymes from the same strain
 For example, HindIII (pronounced “hindee-three”) was
discovered in Haemophilus influenza (strain d).“H”
for Haemophilus
Selection of Restriction Enzymes
Step 1: Identify restriction enzyme sites on your vector by looking at
a restriction map. The restriction map will tell you which enzymes
will cut your vector, and where.
Step 2: Choose a restriction enzyme that also has a site present on
your gene insert, by looking at the sequence of the insert. Ensure
the restriction site is at a position on your insert that is outside the
gene of interest, so you do not lose any part of the gene.
Step 3: Ensure that there are no duplicates of the restriction site
anywhere in your gene insert or vector. This will cause multiple
cuts in your DNA and give you misleading data.
Selection of Restriction Enzymes
Step 4: Try to choose restriction enzymes that cut with sticky ends, rather
than blunt ends. Sticky ends occur when the enzyme cuts double stranded
DNA in a staggered manner, leaving a single stranded overhang that
facilitates attachment with an insert cut in the opposite manner. Blunt
ends occur when the doublestranded DNA is cut in a smooth manner, and
these are more difficult to attach.
Step 5: Choose a different restriction enzyme for both ends of your insert
to ensure it is inserted into the vector in the proper orientation and to
ensure the vector does not re-attach to itself.
Step 6: Try to choose two restriction enzymes which function well in the
same buffer system and temperature. If this is not possible, run each
digestion separately.
Application
 They are used in gene cloning and protein expression experiments
 Restriction enzymes are used in biotechnology to cut DNA into
smaller strands in order to study fragment length differences
among individuals (Restriction Fragment Length Polymorphism-
RFLP)
 Each of these methods depends on the use of agarose gel
electrophoresis for separation of fragments.
 Restriction enzymes are used for DNA sequencing, mutational
analysis, and cloning and amplification of DNA.
 Scientists use restriction enzymes to insert genes of interest into
expression vectors, DNA molecules that can replicate separately
from chromosomal DNA
Ligase
Ligase
 DNA ligases are the ligases that join or bind the two DNA fragments (also
known as Okazaki fragments) by forming a phosphodiester bond.
 The final phosphodiester linkage between the 5’-phosphate group on the DNA
chain synthesized by DNA polymerase III and the 3’-hydroxyl group on the
chain made by DNA polymerase I is catalyzed by DNA ligase.
 The most organism requires energy for the cleavage of ATP and NAD+. Energy
is required for the joining of nicks (nicks lack the phosphodiester linkage
between the nucleotide sequence) of DNA.
 Whereas eukaryotic DNA ligases are ATP-dependent bacteria, archaea and
virus DNA ligases are both NAD+ and ATP dependent.
 DNA ligase forms a bond between the sugar-phosphate backbone to fully
repair the DNA.
 DNA ligase plays a vital role in DNA replication, DNA repair, and DNA
recombination. Now day purified DNA ligase is isolated in the laboratory,
which is used in gene cloning to join DNA molecules together to form
recombinant DNA.
Types of DNA Ligase

ATP dependent DNA ligases:

 Most eukaryotic DNA ligases use ATP as a cofactor,
including bacteriophage, archaeal, and eubacteria DNA
ligases.
 They vary in size in different organisms. The enzyme
found in Haemophilus influenza consists of 268 amino
acids, and larger cellular ligases such as human DNA ligase
I consist of 912 amino acids, and IV consist of 844 amino
acids.
Types of DNA Ligase

NAD+ dependent DNA ligases

 Bacterial DNA ligases are NAD+ dependent ligases. They
are also present in some mimivirus and entomovirus.
While some bacteria, archaea, and viruses, DNA ligases
are both NAD+ and ATP dependent.
 Mycobacterium tuberculosis codes for NAD+ dependent
ligases and at least three different types of ATP-
dependent ligases.
 They have a finely uniform size and consist of 656 – 837
amino acids.
ATP dependent DNA ligases:
Bacteriophage T7:
 It is the most widely used DNA ligase derived from the T7
bacteriophage. It is a monomeric form with a molecular
weight 41KDa.
Bacteriophage T4:
 It can ligate either cohesive end or blunt ends of DNA, RNA
-DNA hybrid, and also RNA.
 It is a monomeric form with a molecular weight 68KDa.
NAD+ dependent DNA ligases
E. coli DNA ligases:
 It is a monomeric enzyme of molecular weight 74KDa and requires
NAD+ as a cofactor.
 It catalyzes the formation of the phosphodiester bond in double-
stranded DNA containing cohesive ends.
 LigA and LigB (product of lig gene) are mostly used E. coli DNA
ligases.
Taq DNA ligases:
 It is a thermostable ligase identified in several thermophilic bacteria,
they require NAD+ as a cofactor.
 It is more stable and active at extreme temperatures than conventional
DNA ligases. It is used in DNA amplification reactions to detect
mutations in mammalian DNA.
Application of Ligase
 DNA replication, DNA repair, and recombinant DNA experiment are incomplete
without DNA ligases. DNA ligases join the nicks and form a phosphodiester
bond between the nucleotide.
 Several laboratories purified DNA ligases from different organisms, which are
used in gene cloning to join DNA molecules together to form recombinant DNA.
 They are used with restriction endonuclease enzyme to insert DNA fragments,
often genes, into the plasmid.
 E. coli DNA ligases are used for high-efficiency cloning of full-length cDNA.
 Commercially available thermostable Taq ligases are used in amplification
reactions because of their thermostable properties.
 Human DNA ligase IV is required for V(D) J recombination, the process that
generates diversity in immunoglobulin and T-cell receptor loci during immune
system development.
Phosphatase
Phosphatase

 A phosphatase is an enzyme that removes a phosphate

group from its substrate by hydrolyzing phosphoric acid
monoesters into a phosphate ion and a molecule with a
free hydroxyl group.
 This action is directly opposite to that of phosphorylases
and kinases, which attach phosphate groups to their
substrates by using energetic molecules like ATP.
Types of Phosphatase

 On the basis of their activity, there are two types of

phosphatase:
 ACID PHOSPHATASE
 ALKALINE PHOSPHATASE
 In both forms, the alkaline phosphate are most common
special class of phosphatase that remove a phosphate
group from protein called phasphoprotein phosphatase
pH Optimum:
 Acid phosphatase (ACP): Functions optimally at a low pH, typically
around 5.0.
 Alkaline phosphatase (ALP): Functions optimally at a high pH, typically
around 9.0.
Source
 Acid phosphatase (ACP): Primarily secreted by plant roots, especially
when phosphorus levels are low, allowing them to access organic
phosphorus in the soil.
 Alkaline phosphatase (ALP): Primarily produced by soil microbes,
contributing to the overall phosphorus cycling in the soil ecosystem.
Kinase
Kinase
 Kinase is an enzyme that catalyzes the transfer of phosphate groups
from high-energy, phosphate-donating molecules to specific
substrates. Kinases are critical in metabolism, cell signalling, protein
regulation, cellular transport, secretory processes, and many other
cellular pathways.
 Eukaryotic organisms possess three main types of Kinases, which are
classified according to the amino acid side chain that they
phosphorylate:
 Tyrosine kinases that phosphorylate the Tyr phenolic hydroxyl
 Serine-Threonine kinases that phosphorylate the hydroxyl group of
two amino acids
 Histidine kinases that phosphorylate the nitrogen of His residues.
Kinase and Phosphatase

 Protein kinases and phosphatases are both

phosphotransferases, however their function are tightly
regulated.
 Phosphorylation is always catalyzed by kinases
 Dephosphorylation is driven by phosphatases.
 Phosphorylation and dephosphorylation are critical for
signaling, cell division, protein translation, metabolism and
survival
 These two processes, phosphorylation and
dephosphorylation, occurs four times during glycolysis.
Transferase
Transferase

 Transferase is the general name for the class of enzymes

that enact the transfer of specific functional groups (e.g. a
methyl or glycosyl group) from one molecule (called the
donor) to another (called the acceptor).
 They are involved in hundreds of different biochemical
pathways throughout biology, and are integral to some of
life’s most important processes.
Terminal Transferases

 Terminal transferases can be used to label DNA or to

produce plasmid vectors.
 It accomplishes both of these tasks by adding
deoxynucleotides in the form of a template to the
downstream end or 3' end of an existing DNA molecule.
 Terminal transferase is one of the few DNA polymerases
that can function without an RNA primer
Polymerase
Polymerase

 A polymerase is an enzyme that synthesizes long

chains or polymers of nucleic acids.
 DNA polymerase and RNA polymerase are used to
assemble DNA and RNA molecules, respectively,
by copying a DNA or RNA template strand using
base pairing interactions.
Prokaryotic DNA Polymerase
1. Polymerase I (Pol I)
 Prokaryotic Family A polymerases include the DNA
polymerase I (Pol I) enzyme, which is encoded by the polA
gene and ubiquitous among prokaryotes.
 This repair polymerase is involved in excision repair with
3'-5' and 5'-3' exonuclease activity and processing of
Okazaki fragments generated during lagging strand
synthesis.
 Pol I is the most abundant polymerase accounting for >95%
of polymerase activity in E. coli
 Pol I adds ~15-20 nucleotides per second, thus showing
poor processivity.
Prokaryotic DNA Polymerase

2. Polymerase II (Pol II)

 DNA polymerase II, a Family B polymerase, is a polB gene product.
 Pol II has 3'-5' exonuclease activity and participates in DNA repair,
replication restart to bypass lesions, and its cell presence can jump
from ~30-50 copies per cell to ~200-300 during SOS induction.
 Pol II is also thought to be a backup to Pol III as it can interact with
holoenzyme proteins and assume a high level of processivity.
 The main role of Pol II is thought to be the ability to direct
polymerase activity at the replication fork and helped stalled Pol III
bypass terminal mismatches.
Prokaryotic DNA Polymerase

3. Polymerase III (Pol III)

 DNA polymerase III holoenzyme is the primary enzyme
involved in DNA replication in E. coli and belongs to
Family C polymerases.
 It consists of three assemblies: the pol III core, the beta
sliding clamp processivity factor and the clamp-loading
complex.
 The core consists of three subunits - α, the polymerase
activity hub, ɛ, exonucleolytic proofreader, and θ, which
may act as a stabilizer for ɛ.
RNA polymerase (RNAP, RNApol, or
DNA-dependent RNA Polymerase
 Eukaryotic RNA polymerase I synthesizes a pre-rRNA 45S
(35S in yeast), which matures into 28S, 18S and 5.8S rRNAs
which will form the major RNA sections of the ribosome.
 Eukaryotic RNA polymerase II synthesizes precursors of
mRNAs and mostsnRNA and microRNAs. This is the most
studied type, and, due to the high level of control
required over transcription, a range of transcription
factors are required for its binding to promoters.
 Eukaryotic RNA polymerase III synthesizes tRNAs, rRNA 5S
and other small RNAs found in the nucleus and cytosol.
Nuclease
Nuclease

 A nuclease is an enzyme capable of cleaving the

phosphodiester bonds between the nucleotide subunits of
nucleic acids.
 Nucleases are usually further divided into endonucleases
and exonucleases, although some of the enzymes may fall
in both categories. Well known nucleases are
deoxyribonuclease and ribonuclease.
Exonucleases
 It catalyses hydrolysis of terminal nucleotides
from the end of DNA or RNA molecule either 5’to 3’
direction or 3’ to 5’ direction.
Endonucleases
 Endonucleases cleave the phosphodiester bonds
within the DNA molecule.
Ribonuclease (RNase)
 Nucleases that can catalyze hydrolysis of ribonucleotides
from either single stranded or double stranded RNA
sequence are called ribonucleotides (RNase).
 They are classified into two types depending on position
of cleavage, i.e. endoribonuclease (cleave internal bond)
and exoribonuclease (cleave terminal bond). RNase is
important for RNA maturation and processing.
 RNAseA is an endo-ribonuclease that cleaves specifically
single-stranded RNA at the 3‘ end of pyrimidine residues.
 RNaseH is a ribonuclease that cleaves the RNA in a
DNA/RNA duplex to produce ssDNA.
Deoxyribonuclease (DNase)
 A nuclease enzyme that can catalyze the hydrolytic
cleavage of phosphodiester bonds in the DNA backbone
are known as deoxyribonuclease (DNase).
 DNase does not have any specific recognition/restriction
site and cleave DNA sequence at random locations.
Deoxyribonuclease I (DNaseI)
 An endonuclease which cleaves double-stranded DNA or
single stranded DNA.
 The cleavage preferentially occurs adjacent to pyrimidine
(C or T) residues.
 Some applications of DNase I are as follows:
1. Eliminating DNA contamination (e.g. plasmid) from
preparations of RNA.
2. Analyzing the DNA-protein interactions via DNA
footprinting.
3. Nicking DNA prior to radio-labeling by nick translation.
Application of Nuclease
 It is used to remove RNA contamination from DNA sample
 Non-specific endoribonuclease that degrades RNA by
hydrolytic mechanism from DNA/RNA duplex resulting in
single stranded DNA.
 Enzyme bound divalent metal ion is a cofactor here. The
product formed is 5’ phosphorylated ssDNA.
 During cDNA library preparation from RNA sample,
RNaseH enzyme is used to cleave RNA strand of DNA-RNA
duplex.