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DNA Structure and Chemistry
Introduction
We will talk today about the structure of DNA ,genes and chromosomes. This
is a very important subject in molecular genetics and in medicine because all genetic
diseases are resulted from changes in DNA , so you have to understand the structure ,
behavior and some general characteristics of DNA . Our DNA is always exposed to
changes from the environment m chemicals , drugs ,food, and many other sources ; so
that means that DNA is exposed tomutations.
Genetic diseases are caused by mutations , so there must be a certain system in
our body to repair all these changes that cause mutations. So in order to understand
how these mutations happen ( to understand the molecular basis of genetic diseases )
you have to understand how mutations take place and how they are repaired ; so you
have to be familiar with the structure of DNA , thus the structure of genes and
chromosomes.
Every somatic cell in our body contains specific number of chromosomes ( 46
chromosomes). And every chromosomes carries a single of a double stranded DNA
molecule ( we have 46 double stranded DNA molecule in total per every somatic cell
). If the DNA molecule is extended , the length of this DNA molecule will be at least 2
meters ; so we have to understand the structure of DNA molecules and how they have
managed to fit in the very small nucleus.
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The mutations of DNA will stop
the flow of the genetic material from
DNA to DNA ( replication) , from
DNA to RNA ( transcription and
processing) and from RNA to protein
synthesis ( translation). This is called
the " Genetic Dogma" (The Central
Dogma of Molecular Biology).
*There is also the flow of genetic material from RNA to DNA which is called "The
Reverse Transcription" which is found in many retroviruses.
Any change in these processes (replication, transcription and translation ) due to
mutations or any change in the structure of DNA will eventually cause " Genetic
Disease".
THE FLOW OF GENETIC INFORMATION
DNA RNA PROTEIN
DNA
1
2 3
1. REPLICATION (DNA SYNTHESIS)2. TRANSCRIPTION (RNA SYNTHESIS)3. TRANSLATION (PROTEIN SYNTHESIS)
The Genetic Dogma : is the flow of genetic material from DNA toDNA, from DNA to RNA and from RNA to protein synthesis.
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DNA Structure
a) Evidence that DNA is the genetic information :Previously , it was believed that protein is the genetic material that transfers
genetic information from one generation to the other. But later they discovered that
protein is not the genetic material ,it's actually DNA. This discovery was achieved by
doing many experiments. One of the most important experiments was " DNA
Transformation".
1) DNA Transformation experiments:
- Objective : prove that DNA is the carrier of the genetic information .
- These experiments have been carries out both in vivo (in animal) and in vitro ( in cell
culture).
- In Vivo :
The experiments were carried out by injecting an animal (mice) with a mixture
of a heat-killed virulent strain of a microorganism ( streptococcus or pneumococcus
with certain genotype and phenotype ) and a nonheat-killed non-virulent of the samestrain .
Note: each of the previous strains is not infectious when injected into the animal
solely.
The experiment showed that something (DNA) from the heat-killed virulent
strain was able to alter the ( still viable) non-heated , non-virulent strain's DNA ,
converting it into virulent bacteria ( transformation ) causing infection to the host
animal . ( The morphology and the behavior of the non-virulent bacteria have beenchanged because the genotype has changed )
-Results of the experiments:
Since all proteins , carbohydrates and lipids were hydrolyzed by heating; so this
indicates that DNA is the genetic material . ( The genetic material of some viruses is
RNA )
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The experiments that prove
DNA as the genetic material
DNA Transformationex eriments
-In Vitro :
This experiment was carried out by mixing 2 strains of pneumococcus bacteria;
Type s ( smooth colony) and Type R (rough colony).
It has showed that the smooth colony was transformed into the rough colony
due to the transfer of DNA from the rough colony to the smooth one changing the
genotype of the smooth colony which resulted in changing the genotype into the
rough colony'scharacteristics.
2) Transgenic experiments :
- Objective : prove that DNA is the carrier of the genetic information .
This experiment is carried out by inserting a foreign DNA or a certain gene into
a fertilized egg which will integrates into the chromosomal DNA of this egg. When
this egg grows and build an organism , this organism will carry the inserted DNA or
gene in all or some of it's cells . so the genotype of this organism will be changed
resulting in a phenotype change as well .
-The animal that has the inserted gene or DNA is called the " transgenic animal".
3) The knockout experiments :
- It's another type of the transgenic experiments that was also designed to prove DNA
as the genetic material .
- This experiment was carried out by the destroying certain genes ( making mutation)
in an animal to determine the function of these genes by observing the phenotype
changes which were definitely caused by the genotype changes ( mutations).
Transgenic experiments The knockout experiments
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Promoters: are regions found
infront of any gene and they are
specific DNA sequences which
are responsible for regulating that
gene expression process.
b) structure of DNA :
we are familiar with the
structure of DNA from the previous
lectures . we talked about thenitrogenous bases ( Adenine,
Guanine, Thymine, Cytosine) and
now we will consider a new base
which is 5-methylcytosine (5mC).
The only difference between
5mC and cytosine is the methyl
group on carbon 5.
DNA gets modified very frequently in our cells after it's synthesis ; methylation
of cytosine is one of these modifications. Cytosine is mostly methylated and that is
after DNA synthesis .
This methylation process is very important in the regulation of gene
expression :-
When a gene is highly methylated it will not be expressed ( inactive)
When a gene is demethylated it will be expressed ( active )
Methylation of cytosine residues is near
promoters is very crucial . within these promoter
regions there are many nucleotide residues
including cytosine .when these cytosine residues in
the promoters get methylated the promoters will be
inactive and thus the gene will not be expressed.
The problem that arises from this methylation process is that when the
methylated cytosine (5mC ) gets deaminated ( one type of DNA modifications) , it
results in the production of thymine .
Thymine (T)
Guanine (G) Cytosine (C)
Adenine (A)
Structures of the bases
Purines Pyrimidines
5-Methylcytosine (5mC)
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Hot islands CG : are the sites where 5mC residues are often
clustered near the promoters of a gene , and there they cause
mutations and thus cancer when they get deaminated .
( CG stands for cytosine and guanine )
We mark the carbons of the sugar
with a prime (C1' , C2' ,C3',) to
distingush them from the carbons
of the N base .
Since thymine is not foreign to DNA . the repair systm of DNA will leavt it
unchanged .this will result in a mutation that will be carried from one generation to the
next , and it will eventually cause cancer. That's why 5-methylcytosine (5mC) is
considered to be a "mutagenic compound".
Considering the structure of a
nucleotide and a nucleoside of a
DNA molecule , you can notice :
-The bond between the nitrogenous
base and the sugar is called
glycosidic bond ; it's formed
between N9(of the N base) and
C1'(of the sugar) .
-A nucleoside is a N base and asugar linked by a glycosidic bond.
-When the phosphate group is attached to the
sugar's C5' the structure is called a
nucleotide.
-The DNA nucleotide's sugar is a deoxyribose;
that lacks a hydroxyl group on C2'.
-The C3' forms a phosphodiester bond with the next nucleotide.
[structure of deoxyadenosine]
Nucleoside
Nucleotide
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This table lists the common
bases and their corresponding
names when in the nucleoside or
nucleotide form. And you have to
be familiar with them.
The structure of the DNA polynucleotide chain
-DNA is an anti-parallel
double-stranded
polynucleotide chain.
-Each strand is
complementarily base-paired
to the other.
- It has a helical structure
due to the chemical nature of
the N bases ( the helical
structure helps DNA to fit
inside the small nucleus).
- Each DNA polynucleotide chain has a beginning ( 5' end) and an ending ( 3'
end)
(the sequence of any gene always runs from 5' to 3' )
- 5' end always has 3 phosphate groups .
-3'end has a 3' hydroxyl group .
Nomenclature
Purines
adenine adenosineguanine guanosine
hypoxanthine inosine
Pyrimidines
thymine thymidinecytosine cytidine
+ribose
uracil uridine
Nucleoside Nucleotide
Base +deoxyribose +phosphate
polynucleotide chain 3,5-phosphodiester bond
ii). Structure of theDNA double helix
Structure of the DNA
polynucleotide chain
5
3
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The resonance phenomena : is
the redistribution of electrons on
the surface of the molecule
between atoms
- Every two nucleotides are linked
via 3',5'-phosphodiester bond .
- The two DNA strands are base-
paired in a complementary way:
Specific purines from one strand
will base-pair with a specific
pyrimidine in the other strand.
According to "Chargaff Rule" , Adenine ( purine) always base-pairs with
Thymine (pyrimidine) and Guanine ( purine) always base-pairs with cytosine (
pyrimidine). The Chargaff rule was fulfilled when Watson and Crick discovered
the double stranded DNA structure .
These complementary bases are linked together via hydrogen bonds .
( You have to know the numbers of atoms involved in the hydrogen bonding in
the nitrogenous bases )
These hydrogen bonds between
complementary bases can be changed
according to the "resonance phenomena" of
these purine and pyrimidine rings, and thesechanges could cause mutations .
This resonance phenomena will disrupt
the hydrogen bonds; so instead of having A paired with T, we will have A paired
with C for example , and this will cause mutation .
A-T base pair
G-C base pair
Chargaffs rule: The content of A equals the content of T,
and the content of G equals the content of Cin double-stranded DNA from any species
Hydrogen bonding of the bases
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DNA is originally compact when
it's not expressed, when proteins
bind to it , DNA will unwrap to
be exposed to all other proteins
for DNA ex ression .
*Why dose DNA exist in a double- stranded form ?
It's very important for DNA to be double- stranded, and to be composed of two
base-paired , complementary and anti-parallel polynucleotide chain , and that is
required for many functions of DNA :
1)DNA replication :
If DNA was a single polynucleotide chain, the cell will not be able to synthesis
a new identical DNA molecule . In DNA replication each strand of DNA
molecule serves as a template for the complementary synthesis of the second
strand after the unwinding of the double helix has occurred .
2)DNA repair mechanism :If DNA was composed of one strand and one nucleotide was deleted , the repair
system of DNA won't be able to correct it because the cell's repair system
requires a template; in order to read it and then insert the complementary proper
deoxynucleotide. So DNA must be a double stranded polynucleotide.
DNA molecule can exist in many forms ( A form , B form , Z form) ,butour concern will be DNA in the B form which has many characteristics :
- Major grooves and minor groovesFor a gene to be
expressed and protein to be
synthesized , many proteins (
e.g. Transcriptional factors)
should interact with that genein order to activate it or
sometimes suppress it. Those
proteins bind very specifically
to certain sites on genes, and
these sites are called " major
grooves".
Double-stranded DNA
Major groove
Minor groove
5 3
5 33 5
B DNA
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These proteins then start scanning and reading the sequence of nucleotides in
the major grooves. ( proteins can read the sequence of nucleotides and bind to them
ONLY in the major grooves ;because major grooves are exposed to them while minorgrooves are not exposed )
- It has 10 base pairs per turn of the double helix , and the distance betweenpairs is 3.4 A .
*supercoiled DNA:
-when DNA molecule has 10 base pairs / turn, it's said to be in the
relaxed supercoil .
-when it has more than 10 base pairs/ turn ( over winding) , it's called
positive supercoil .
-when it has less than 10 base pairs / turn (under winding) , it's called
negative supercoil. ( required during replication and transcription )
During replication and transcription , positive supercoil will be formingin one end (restricted ) while the two strands are unwinding ( opening) at the other
end ( negative supercoil is forming here ) .
As the unwinding continues at one end , the positive supercoil will
increase to reach a high degree at the other end of the DNA molecule .so the two
strands will resist further unwinding that could result in preventing the unwinding,
DNA replication, transcription and gene expression .
supercoiled DNA
positive supercoil
>10 base pairs / turn
negative supercoil
< 10 base airs turn
relaxed supercoil
= 10 base pairs / turn
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The cell has certain mechanisms that convert the positive supercoil into
relaxed or negative supercoil ; in order to let the two strands continue unwinding to
complete the replication , Transcription and thus gene expression.
*Nucleases :
Nucleases are enzymes that hydrolyze (cleave) the phosphodiester bonds,
and they are of two classes :
1)Endonucleases : cleave nucleotides anywhere within the DNA double helix from
5'-3' or 3'-5' direction .
2)Exonucleases : cleave only terminal nucleotides from 5' end or 3' end .
These enzymes are important tools in genetic engineering and for constructive
purposes such as proofreading during DNA replication.
Chemistry of DNA
*How the DNA double-helix structure is stabilized?
1)Hydrophobic interaction : ( by N bases , stabilizes )
Recall that N bases are organized inside
the double helix in linear form and they are on
top of each other ( imagine a cylinder with
coins inside it on top of each other). Because of
the chemical nature of the purines and
pyramidines this kind of arrangement of the N
bases will create a hydrophobic interaction that
will stabilize the double helical structure .
2) Stacking interaction : ( by N bases , stabilizes )
This type of interaction is weak but
additive .
3) Hydrogen bonding : ( by N bases , stabilizes )
Complementary N bases forms hydrogen
bonds. Although these hydrogen bonds are weak
Stacking interactions
Charge repulsion
Chargerepulsion
Model of double-stranded DNA showing three base pairs
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but additive as they are tremendous in number ; so they will form a strong force that
will stabilize the helical structure .
4) Electrostatic interaction : ( by sugar-phosphate backbone , destabilizes)
The phosphate group of the phosphate-sugar backbone of each strand carries a
negative charges, and these negative charges form whats known as the inter and
intra repulsive forces , and that will destabilize the double helix.
Positive ions ( Na+) and positively charged proteins ( histons and protamines)
bind to the negative charges of the phosphate groups and stabilize the double helix
structure .
Denaturation of DNA
- DNA denaturation is simply the
separation of the two strands by
overcoming the forces that stabilize theDNA double helix .( this denaturation is
important in DNA replication )
-These stabilizing forces can be
overcome by enzymes or by heating the
DNA ( DNA is then said to be melted)
and that will disrupt the hydrogen
bonding.
Histones : are small basic proteins, important for expression and
stabilizing the double helix because of the positive-negative
interaction.
Why they are positively charged ?
Because they are rich with lys and Arg amino acids which are
positively sharged at phsyological p H.
Denaturation of DNA
Double-stranded DNA
A-T rich regionsdenature first
Cooperative unwindingof the DNA strands
Extremes in pH orhigh temperature
Strand separation
and formation ofsingle-strandedrandom coils
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-The stabilizing forces can be also overcome by very high or very low pH , and this
will dissolve N bases.
- Low pH will also damage the DNA . whereas high pH will simply separate the two
strands .
When the DNA molecule starts
to partially denature ( separate ) you
will notice that there are some regions
that have not separated yet; and that
depends on the structure and
composition of the DNA molecule.
The regions that havedenatured first upon exposure to heat
, pH or any other factor , they are
rich in A-T base pair .while other
regions which are rich in the base pair
G-C will denature later. This
difference in t he timing of
denaturation is due to the strength of the hydrogen bonding between A-T and G-C .(
as you know that G-C pair has three H bonds while A-T pair has only two H bonds )
The unwinding in DNA denaturation is cooperative; meaning that there will be
a resistance in order to denature the first region, and after the DNA molecule acquires
more energy by heat or any other factor , the next unwinding will be easier .
*can we reverse denaturation ?
Yes we can( renaturation) and it depends on the size of the DNA molecule .
Electron micrograph of partially melted DNA
A-T rich regions melt first, followed by G-C rich regions
Double-stranded, G-C richDNA has not yet melted
A-T rich region of DNAhas melted into asingle-stranded bubble
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Hyperchromicity :
*What is hayperchromicity of DNA?
It's a phenomena which indicates
that denaturized DNA molecule ( single
stranded DNA) will absorb more light
than renatured DNA molecule ( double
stranded DNA) at the same wave length.
Why is that ?
Because the single stranded DNA has more surface area exposed to light than
the double stranded DNA; as the N bases ( which absorb the light) are much more
exposed to light in the single stranded DNA than in the double stranded one .
Hyperchromicity is important in studying the melting curve of DNA
( denaturaion of DNA by heat ) and this will be discussed next lecture .
The End
Done by
Rinad Al-Ali
Hyperchromicity
The absorbance at 260 nm of a DNA solution increaseswhen the double helix is melted into single strands.
260
Absorbance
Absorbance maximumfor single-stranded DNA
Absorbancemaximum fordouble-stranded DNA
220 300