genetics lec #3 - dna structure and chemistry - by rinad alali

8/3/2019 Genetics Lec #3 - DNA Structure and Chemistry - By Rinad Alali

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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

genetics lec #3 - dna structure and chemistry - by rinad alali

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