PAPER 0703

Basics Concepts and Techniques in Molecular

Biology

Lecture 2
(Semester I-2015)
OUTLINE OF TODAY’S LECTURE

 Nucleic Acid Manipulating Enzymes

 Nucleases
 deoxyribonucleases
 ribonucleases

 Ligases

 Polymerases
Terminal Deoxynucleotidyl transferase

 Nucleic Acid Modifying

Enzymes

 Alkaline Phosphatase

 Polynucleotide Kinase
 LIGASES

Catalyze formation of a phosphodiester bond between adjacent 5’-P

and 3’-OH termini.

DNA Ligases RNA Ligases

E. coli DNA ligase T4 RNA ligase

T4 DNA ligase

Taq or Pfu DNA ligase

 Mechanism of Ligation

1. Formation of an adenylate-enzyme complex involving either ATP or NAD.

2. Release of pyrophosphate and AMP residue is bound covalently to amino-group of

lysine residue in the enzyme via a phosphoric acid amide bond.

3. 5’ PO4 residue is activated by transfer of this adenylate residue.

4. Nucleophilic substitution of 3’-OH substituent at the activated 5’-PO4 residue.

5. Formation of the phopshodiester bond accompanied by release of AMP.

Mechanism of Ligation
I. Physical reaction: DNA end
collide in a solution.

!!. Enzymatic reaction:

Formation of adenylate complex with

the enzyme (enzyme-AMP).
Activation of enzyme by covalent
protein-AMP intermediate an release
of Ppi/NMN.

AMP nucleotide (attached to

lysine residue in enzymes
active site) is transferred to the
5’-phosphate of the nick to produce
5-5’ pyrophosphate bridge structure.

Transesterification reaction resulting

in joining of nicks. AMP-phosphate
bond is attacked by 3’-OH, forming
the covalent bond and release of
AMP.

AMP needs to be replenished in the

enzymes active site by ATP in
 DNA LIGASES

E. coli DNA Ligases

♣ Biological Source: E. coli
♣ Catalyzes a phosphodiester bond between duplex DNA containing cohesive ends.

♣ Does not efficiently ligate blunt ended fragments.

♣ Requires NAD+ as a cofactor.
 DNA LIGASES

T4 DNA Ligases
♣ Biological Source: T4 bacteriophage
♣ Catalyzes a phosphodiester bond between
Duplex DNA containing cohesive ends.
Duplex DNA containing blunt ends
Joins nicks in RNA chains of ds DNA-RNA
hybrids
Anneals RNA termini with DNA strands

♣ Requires ATP as a cofactor. .

 DNA LIGASES

Taq or Pfu DNA Ligases

♣ Biological Source: Thermus or Pyrococcus
♣ catalyze a phosphodiester bond between two adjacent
oligonucleotides which are hybridized to a complementary
DNA strand.

♣ efficient only if the oligonucleotides hybridize perfectly with the template strand.
♣ enzyme is active at relatively high temperatures (45 - 65 °C).
♣ requires NAD+ as a cofactor.
 RNA LIGASES

♣ Catalyzes formation of a phosphodiester bond between RNA/RNA oligonucleotides,

RNA/DNA oligonucleotides, or
DNA/DNA oligonucleotides.
♣ Requires ATP as a cofactor.
♣ Does not require a template strand or complementary strand.
♣ Employed in a variety of purposes including constructing RNA/DNA hybrid molecules.
 Factors Affecting Ligase Activity

1. Substrate Specificity

♣ Ligation activity is not influenced by the sequence.

♣ Different enzymes have specificity for different kinds of DNA or RNA molecules.
♣ Specificity for substrate A, B & C can be switched to A & B by increasing ATP concentrations
from 0.5 mM to 5 mM.

High concentrations of ATP causes reversible inhibition of blunt-end ligation activity of

T4 DNA ligase.

Increasing ATP conc. to 7.5 mM reversibly inhibits both activities.

Appropriate ATP conc. can be employed for ligating both kinds of ends sequentially in
the same reaction mix.

A B C
 Factors Affecting Ligase Activity

2. Temperature

♣ Optimal ligation temperature is 37º C.

♣ At 37º C base pairing between complementary protruding ends is unstable.

♣ Compromise for a favorable temperature is between optimal enzyme activity and optimal base pairing.

37º C 16 or 25º C
Ligase Activity—High Ligase Activity—Lower

Base pairing------Low Base pairing------High

(stability) (stability)

Trade-ff between the optimal temperature for bringing the DNA ends together
and the enzymatic reaction
 Factors Affecting Ligase Activity

3. Concentration

♣ Bimolecular reactions between vector and insert are desirable.

♣ Intra- and inter-molecular reactions do occur.
♣ Concentration of vector and insert molecules be adjusted so that bimolecular reactions are favored.

PEG may be added to enhance ligation efficiency

 ALKALINE PHOSPHATASE

♣ Catalyzes removal of 5’-phosphate groups from DNA, RNA

♣ CIP-treated fragments lack 5’-phosphoryl termini, they cannot self-ligate
♣ Useful in cloning as employed in decreasing vector background
♣ Isolated from Calf intestine (CIAP) or Shrimp (SAP)
♣ Hemi-phosphorylated duplexes will be ligated on one strand (the phosphorylated strand) and
remain "nicked" on the other.
Cloning Strategy Using Alkaline Phosphatase
 POLYMERASES

Catalyze formation of a new strand of DNA or RNA

DNA Polymerases RNA Polymerases

E. coli DNA Polymerase I

Klenow Fragment

Klenow Fragment (3’ 5’) exo -

T4 DNA Polymerase

Terminal transferase

Reverse Transcriptase

Thermostable DNA Polymerase

Properties Of DNA Polymerases
Generalized Structure of DNA Polymerase

Nuclease
 Activities Of DNA Polymerases

Three main activities:

1. PROCESSIVITY
or
5’ 3’ Polymerase
activity.
 Activities Of DNA Polymerases

2. 3’ 5’ Exonuclease activity- allows the polymerase to correct a mistake if it incorporates

an incorrect nucleotide (so called "error correction activity").

Can also slowly degrade the 3' end of the primer.

 Activities Of DNA Polymerases

3. 5’  3’ Exonuclease activity- allow it to degrade any other hybridized primer it may

encounter.

Without 5'->3' exonuclease activity, obstructing primers may or may not be physically
displaced, depending on the polymerase being used.

Activities of DNA Polymerase Affected by pH conditions

pH 7.0 -------------> Polymerase is 2X higher than nuclease

pH 8.6 ------------ Exonuclease is 4X active than polymerase

 Terminal Deoxynucleotidyl Transferases

♣ Isolated from calf, rat or mouse thymus.

♣ Does not require a template (template independent).
♣ Requires at least three nucleotides to serve as the primer.
♣ Protruding, recessed or blunt-ended double or single-stranded DNA molecules as
substrates.
♣ Preferred substrate is 3’-OH end of protruding DNA end.
♣ DNA molecules with blunt ends or 5’ protruding ends are poor substrates.
♣ Optimization of reaction conditions such as Co2+ ions----dA & dT
MnCl2---------dC & dG.

♣ Repetitive addition of deoxyribonucleotides to the 3’-OH of DNA.

♣ RNA can be also used a substrate but variable performance depending upon the tertiary
structure of acceptor RNA 3’ end and the nature of nucleotide.
♣ Useful in homopolymer tailing of linear duplex DNA & labeling of 3’ ends.
 Applications of DNA Polymerases

1. DNA Polymerase I
5’ 3’ polymerase
♣ Labeling of DNA by Nick Translation in conjunction 3’5’ exo : ++
with DNAse.
5’3’ exo : +

♣ Second strand synthesis of cDNA in conjunction

with Rnase H.
Nick translation as catalyzed by DNA Polymerase
I
 Applications of DNA Polymerases

2. Klenow Fragment

♣ Blunting of 3’ overhang 5’ 3’ polymerase

3’5’ exo : ++
♣ Filling-in of 5’ overhang (labeling) 5’3’ exo : --

♣ Blunting of 3’ overhang (cloning)

♣ Random Priming Labeling
♣ Second strand synthesis of cDNA
♣3’DNA Sequencing 5’ 3’ 5’

5’ 3’ 5’ 3’
 Applications of DNA Polymerases

3. Klenow Fragment exo--

♣ Labeling by fill-in of 5’-overhangs 5’ 3’ polymerase

3’5’ exo : --
♣Random Priming Labeling 5’3’ exo : --

♣ Second strand synthesis of cDNA

♣ DNA Sequencing
4. T4 DNA Ploymerase

♣ Stronger exo activity on ss than on ds DNA and greater than 5’ 3’ polymerase
DNAPol I and Klenow. 3’5’ exo : +++++
5’3’ exo : --
♣ Blunting of 5’(fill-in) or 3’ protruding DNA termini

♣ DNA Labeling
♣ Oligo-based site-directed mutagenesis
 Applications of DNA Polymerases
5. phi29 DNA Ploymerase 5’ 3’ polymerase
♣ Highly processive polymerase (up to 70 kb) 3’5’ exo

♣Strong displacement activity which allows for highly efficient isothermal DNA amplification.
♣ Acts preferentially on ss DNA or RNA.
♣ Multiple displacement amplification.
♣ Unbiased amplification of whole genome (WGA). Very useful in amplification of DNA for
SNP detection or cell-free amplification of DNA from single cells or pathogenic organisms
or metagenomes or even blood samples.

Useful for preparation of template for

sequencing.
Random hexamers bind to DNA
Polymerization
Strand displacement
New primers bind to newly formed DNA
Polymerization begins on new strands
 Applications of DNA Polymerases
6. Terminal Transferase
3’5’ exo : --
♣ Homopolymeric tailing of linear duplex DNA with 3’-OH terminus. 5’3’ exo : --

♣ Oligonucleotide and DNA labeling

♣ In situ localization of apoptosis

 TUNEL Assay for Detection of Apoptosis
Terminal deoxynuleotidyl transferase dUTP Nick End Labeling

DNA fragmentation is a hallmark of apoptosis.

The enzyme adds dUTPs wherever nicks are present.
Can also be employed for assessing DNA damage in cells/tissues.
dUTPs are labeled with a marker. Labeled with fluorophores or biotin.
 Applications of DNA Polymerases

Use of homopolymeric poly (C) tails for the reconstitution of a restriction site
such as Eco RI
 Applications of DNA Polymerases

Use of homopolymeric poly (C) and poly (G) tails for the reconstitution of a restriction
site such as Pst I.
 DNA POLYMERASES

Taq DNA Polymerase

Thermostable Polymerase
Useful in PCR amplification or end-filling

Fidelity improved in DNA Pol from Pfu polymerases, Phusion polymerase

Table 6–1 Error Rates in DNA synthesis

If an incorrect nucleotide is added to a growing strand, the DNA

polymerase will cleave it from the strand and replace it with the correct
nucleotide before continuing.
Pfu amplified fragments are blunt-ended as compared to
Taq generates A overhangs
Comparison Of Fidelity Of Different DNA Polymerases
Fidelity Of Pfu DNA Polymerase is Affected by pH

-- Increase the input template DNA

-- Minimize the denaturation time.
 Polynucleotide Kinase

♣ Catalyzes the transfer and exchange of a phosphate group from the γ position
of ATP to the 5’ -OH terminus of double stranded and single stranded DNA or
RNA, and nucleoside 3' monophosphates (Forward reaction). Reaction is
reversible.

♣ In the presence of ADP, T4PNK exhibits 5’-phosphatase activity and catalyzes

the exchange of phosphate groups between 5’-oligo-polymnucleotides and
ATP (Exchange reaction).

♣ Also possesses 3’ phosphatase for 3’ termini (removes 3’-phosphoryl group

from 3’-phosphoryl polynucleotides, deoxynucleoside 3’-monophosphates and
deoxynucleoside 3’-diphosphates.
 Polynucleotide Kinase

♣ Useful in labeling 5’ termini of DNA or RNA by kinase or exchange reactions.

For eg. Preparing probes.

♣ Useful in addition of 5’ phosphates to synthetic oligonucleotides or DNA or

RNA for ligation.

♣ Phosphorylation of PCR primers.

♣ Removal of 3’-phosphate groups.

♣ T4PNK (3’ phosphatase minus) is also available which can be used for 5’
phosphorylation of 3’ phosphorylated mononucleotides (pNp) to generate a
substrate that can be added to the 3’ end of DNA or RNA and also for 5’ end
labeling of 3’phopshorylated oligos.
Oligonucleotides which are obtained from automated synthesizers lack a 5' phosphate
group, and thus, cannot be ligated to other polynucleotides.
 SUGGESTED READING

 Gene Cloning- T.A. Brown

 Principles of Gene manipulation and genomics- Primrose and Twyman

 From Genes to Clone- Winnacker