• It is a technique that predicts the interaction between a macromolecules and a chemical molecule. • Most of the existing efforts to identify the binding sites in protein-protein interaction are based on analyzing the differences between interface residues and non-interface residues, often through the use of machine learning or statistical methods. • Its major application is to Identify the protein ligand binding sites is an important process in drug discovery and structure based drug design. • Earlier, detecting protein ligand binding site is expensive and time consuming by traditional experimental method. Hence, computational approches provide many effective strategies to deal with this issue.

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GOVERNMENT NAGARJUNA P.G. COLLEGE
OF SCIENCE RAIPUR (C.G.)
M.Sc. MICROBIOLOGY
SEMESTER - 2
2022-23
WRITE UP ON
IDENTIFICATION OF PROTEIN BINDING SITE ON
DNA
SUBMITTED BY :- SUBMITTED TO:-
SNEHA AGRAWAL DEPT.OF MICROBIOLOGY

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INDEX
 ABOUT DNA
 HISTORY
 TYPES OF DNA
 PROTEIN BINDING SITES
 IDENTIFICATION OF PROTEIN BINDING SITE
 REFERENCE

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ABOUT DNA
DNA stands for Deoxyribonucleic Acid , which is a molecule that
contains the instructions an organism needs to develop, live and
reproduce. It is a nucleic acid and is one of the four major types of
macromolecules that are known to be essential for all forms of life.
FEATURES OF DNA
 DNA is a double-stranded helix. That is each DNA molecule is
comprised of two biopolymer strands coiling around each other to
form a double helix structure. These two DNA strands are called
polynucleotides, as they are made of simpler monomer units called
nucleotides.
 Each strand has a 5′end (with a phosphate group) and a 3′end
(with a hydroxyl group).
 The strands are antiparallel, meaning that one strand runs in a
5′to 3′direction, while the other strand runs in a 3′ to 5′ direction.
 The two strands are held together by hydrogen bonds and are
complimentary to each other.
 The deoxyribonucleotides are linked together by 3′ –
5′phosphodiester bonds.
 The nitrogenous bases that compose the deoxyribonucleotides
include adenine, cytosine, thymine, and guanine.
 The complimentary of the strands are due to the nature of the
nitrogenous bases. The base adenine always interacts with a
thymine (A-T) on the opposite strand via two hydrogen bonds and

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cytosine always interacts with guanine (C-G) via three hydrogen
bonds on the opposite strand.
HISTORY
The discovery of DNA’s double-helix structure is credited to the
researchers JAMES WATSON & FRANCIS CRICK . They
presented a model for the DNA’s named as double-helix structure.
Types of DNA
1. B-DNA
Most common, originally deduced from X-ray diffraction of sodium
salt of DNA fibres at 92% relative humidity. This is the most
common DNA conformation and is a right-handed helix. Majority
of DNA has a B type conformation under normal physiological
conditions.
2. A-DNA
Originally identified by X-ray diffraction of analysis of DNA fibres
at 75% relative humidity. It is a right-handed double helix similar to
the B-DNA form. Dehydrated DNA takes an A form that protects
the DNA during extreme condition such as desiccation. Protein
binding also removes the solvent from DNA and the DNA takes an
A form.
3. Z-DNA
Left handed double helical structure winds to the left in a zig- zag
pattern. Z-DNA is a left-handed DNA where the double helix winds
to the left in a zig-zag pattern. It was discovered by Andres Wang
and Alexander Rich. It is found ahead of the start site of a gene and
hence, is believed to play some role in gene regulation.

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DNA BINDING DOMAINS
The transcription factor that interacts with an upstream or response
element. It has two basic domains a dna-binding domain specifically
recognizes the target sequenc -an activation domain contacts a basal
transcription factor.
DNA-Binding proteins serve two principal functions :-
1. To organize and compact the chromosomal DNA.
2. To regulate and effect the processes of transcription, DNA
replication, and DNA recombination.
DNA-binding proteins have the specific or general affinity for single
or double stranded dna by help of DNA-binding domain(DBD).
A DBD can recognize a specific dna sequence or have a general
affinity to DNA. Sequence specific DNA binding proteins generally
interact with major groove of B-DNA because it exposes more
functional groups that identify a base pair.
The transcription factors which modulate process of transcription,
various polymerases, nucleases which cleave DNA molecules
involving in chromosome packaging. DNA binding proteins are :-
1. Non-Histone
2. Histone
There are several motifs present which are involved in DNA binding
that facilitate binding to nucleic acid such as :-
1. Helix turn helix
2. Zinc fingers
3. Leucine zippers
4. Helix loop helix

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IDENTIFICATION OF PROTEIN BINDING SITE
• It is a technique that predicts the interaction between a
macromolecules and a chemical molecule.
• Most of the existing efforts to identify the binding sites in
protein-protein interaction are based on analyzing the
differences between interface residues and non-interface
residues, often through the use of machine learning or
statistical methods.
• Its major application is to Identify the protein ligand binding
sites is an important process in drug discovery and structure
based drug design.
• Earlier, detecting protein ligand binding site is expensive and
time consuming by traditional experimental method. Hence,
computational approches provide many effective strategies to
deal with this issue.
ADVANTAGES OF IDENTIFICATION OF PROTEING
BINDING SITE
1. It quantifies the interaction by calculating binding energies.
2. It provides 3-D information such as orientation of binding.
There are many vitro and vivo techniques which are useful in
detecting DNA protein interactions such as :-

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A. ELECTROPHORECTIC MOBILITY SHIFT ASSAY
B. CHROMATIN IMMUNOPRECIPITATION
C. DNase FOOTPRINTING ASSAY
A. ELECTROPHORECTIC MOBILITY SHIFT ASSAY
An Electrophoretic mobility shift assay (EMSA) or mobility shift
electrophoresis, also referred as a gel shift assay, gel mobility shift
assay, band shift assay, or gel retardation assay, is a
common affinity electrophoresis technique used to study protein
DNA or protein RNA interactions. It is based on the electrophoretic
mobility of a protein- nucleic acid complex.
Mobility-shift assays are often used for qualitative purposes. Under
appropriate conditions they can provide quantitative data for the
determination of binding stoichiometries. affinities and kinetics.
A gel electrophoresis method for quantifying the binding of proteins
to specific DNA regions: application to components of the
Escherichia coli lactose operon regulatory system.
HISTORY
It is discovered by GARNER AND REVZIN AND FRIED AND
CROTHERS.

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PRINCIPLE
It is based on the observations that the electrophoretic mobility of
a protein-nucleic acid complex is typically less than that of the
free nucleic acid.
Smaller molecule of DNA – migrates faster
Larger molecule of DNA – migrates slower
STEPS
1. PREPARATION OF CELL PROTEIN EXTRACT :- purified
proteins, nuclear or cell extract preparations.
2. PREPARE RADIOACTIVELY LABELLED DNA :- Protein is
mixed with radiolabeled DNA Containing a Binding site for
protein.
3. BINDING REACTION :- DNA not mixed with protein runs as
a single band corresponding to the size of DNA fragment. In
the mixture with the protein, a proposition of a DNA molecule
binds the protein. A free DNA band as well as band for DNA-
Protein complex.
4. NON DENATURING GEL ELECTROPHORESIS
5. DETECTION OF OUTCOME :- The mixture is resolved by
PAGE and visualized using autoradiography.

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ADVANTAGES DISADVANTAGES
The mobility shift assay has a
number of strengths
It doesn’t provide a straight
forward measure of the weights
and actual sequences of the
proteins.
The most significant benefit of
EMSA is its ability to resolve
complexes of different
stoichiometry or conformation.
Dissociation is one of the
drawbacks of EMSA. It is not a
appropriate method for kinetic
studies.
Permitting even labile complexes
to be resolved and analyzed by
this method.
Samples are not at chemical
equilibrium during
electrophoresis.
It is a simple,rapid & sensitive
method to perform.
Low cost
It is Quick and versatile method.
B. CHROMATIN IMMUNOPRECIPITATION
Chromatin immunoprecipitation (ChIP) is a type of
immunoprecipitation experimental technique used to investigate the
interaction between the proteins and DNA.
It aims to determine whether specific proteins are associated with
specific genomic regions, such as transcription
factors on promoters or other DNA binding sites, and possibly
define cistromes. ChIP also aims to determine the specific location
in the genome that various histone modifications are associated
with, indicating the target of the histone modifiers. ChIP is crucial

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for the advancements in the field of epigenomics and learning more
about epigenetic phenomena.
HISTORY
It is discovered by JOHN T. LIS & DAVID GILMOUR IN 1984.
PRINCIPLE
The principle behind ChIP is relatively straightforward and relies
on the use of an antibody to isolate, or precipitate, a certain protein,
histone, transcription factor, or cofactor and its bound chromatin
from a protein mixture that was extracted from cells or tissues.
Hence, the name of the technique: Chromatin Immunoprecipitation.
In ChIP-PCR or ChIP-seq, immune-enriched DNA fragments are
then able to be identified and quantified using widely available PCR
or qPCR reagents and Next Generation Sequencing (NGS)
technologies.
STEPS
1. CROSSLINKING :- DNA and associated proteins
on chromatin in living cells or tissues are crosslinked .
2. CELL LYSIS :- The DNA-protein complexes (chromatin-
protein) are then sheared into ~500 bp DNA fragments
by sonication or nuclease digestion.
3. CHROMATIN PREPARATION :- Cross-linked DNA
fragments associated with the protein(s) of interest are

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selectively immunoprecipitated from the cell debris using an
appropriate protein-specific antibody.
4. IMMUNOPRECIPITATATION
5. REVERSAL OF CROSSLINKING AND DNA CLEAN UP
AND DNA QUANTITATION :- The associated DNA
fragments are purified and their sequence is determined.
Enrichment of specific DNA sequences represents regions on
the genome that the protein of interest is associated with in
vivo.
ADVANTAGES DIS ADVANTAGES
Efficient precipitation of DNA
and protein.
• Good for non-histone
proteins binding weakly or
indirectly to DNA. May be
inefficient antibody binding
due to epitope disruption
High resolution (175
bp/monosomes).
Nucleosomes may rearrange
during digestion.
Good for non-histone proteins
binding weakly or indirectly to
DNA. May be inefficient
antibody binding due to epitope
disruption
May be inefficient antibody
binding due to epitope
disruption.
Suitable for transcriptional
factors, or any other weakly
binding chromatin associated
proteins.
Applicable to any organisms
where native protein is hard to
prepare
May cause false positive result
due to fixation of transient
proteins to chromatin
Wide range of chromatin
shearing size due to random cut
by sonication.

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C. DNase FOOTPRINTING ASSAY
A DNase footprinting assay is a DNA footprinting technique
from molecular biology/biochemistry that detects DNA-
protein interaction using the fact that a protein bound to DNA will
often protect that DNA from enzymatic cleavage. This makes it
possible to locate a protein binding site on a particular DNA
molecule. The method uses an enzyme, deoxyribonuclease (DNase,
for short), to cut the radioactively end-labeled DNA, followed by gel
electrophoresis to detect the resulting cleavage pattern.
DNase I footprint of a protein binding to a radiolabelled DNA
fragment. Lanes "GA" and "TC" are Maxam-Gilbert chemical
sequencing lanes, see DNA Sequencing. The lane labelled
"control" is for quality control purposes and contains the DNA
fragment but not treated with DNaseI.
For example, the DNA fragment of interest may be PCR amplified
using a 32
P 5' labeled primer, with the result being many DNA
molecules with a radioactive label on one end of one strand of each
double stranded molecule. Cleavage by DNase will produce
fragments. The fragments which are smaller with respect to the 32
P-
labelled end will appear further on the gel than the longer
fragments. The gel is then used to expose a special photographic
film.
The cleavage pattern of the DNA in the absence of a DNA binding
protein, typically referred to as free DNA, is compared to the
cleavage pattern of DNA in the presence of a DNA binding protein.
If the protein binds DNA, the binding site is protected from
enzymatic cleavage. This protection will result in a clear area on the
gel which is referred to as the "footprint".
HISTORY

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This technique was developed by David Galas and Albert Schmitz at
Geneva in 1977.
PRINCIPLE
In this technique, nucleases like DNAse I is used which will
degrade DNA molecule. Nucleases cannot degrade DNA if it is
bounded by a protein. Thus that region is protected from
degradation by nucleases. This protected DNA region is called the
foot print.
STEPS
1. PREPARE END LABELELD DNA :- Radioactive 5' end
labeling of the DNA suspected to contain one or more protein
binding sites. The DNA is treated with a nuclease such as DNAse
I, that digests only unprotected DNA.
2. BIND PROTEIN
3. MILD DIGESTION WITH DNAASE 1 :- DNAse I is used
under specific digestion condition to obtain one cut or hit per
molecule, resulting in a complete base ladder (one base
difference) when electrophoresed in 6-8% polyacrylamide gel.
4. SEPARATES DNA FRAGMENTS ON GEL :- The resulting
products are separated on a Polyacrylamide gel
electrophoresis (PAGE) In DNA sample with protein, protein
binding regions are protected from degradation by DNAse 5.X-
ray film exposure and autoradiography. Comparison of both
samples reveals foot prints or protein binding sites.

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ADVANTAGES DISADVANTAGES
Powerful enough to differentiate
many fragments.
The enzyme does not cut DNA
randomly
It can exactly locate the binding
site of the particular ligand.
Its activity is affected by local
DNA structure and sequence and
therefore results in an uneven
ladder.
Large foot print can suggest that
two or more proteins bind
adjacent elements on the probe.
Sometimes causes cell
disruption.
It is generally used for scanning
a large DNA fragments or
protein DNA interaction.
REFERENCE
 https://www.creativebiomart.net/resource/principle-protocol-
protein-interaction-4-yeast-one-hybrid-assay-374.htm
 https://www.creativebiomart.net/resource/principle-protocol-
dnase-i-footprinting-
377.htm#:~:text=DNase%20I%20footprinting%20assay%20is,
the%20target%20protein%20is%20bound
 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2757439/
 https://youtu.be/kWeg-5FRqlk
 https://en.m.wikipedia.org/wiki/Chromatin_immunoprecipitati
on

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