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 result 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, chemicals, drugs, food, and many other sources; so that means that DNA is exposed to mutations. 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 a specific number of chromosomes (46 chromosomes). And every chromosome carries a single of a double stranded DNA molecule (we have 46 double stranded DNA molecules 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.

THE FLOW OF GENETIC INFORMATION

2 DNA 1 DNA RNA

3 PROTEIN

1. REPLICATION (DNA SYNTHESIS) 2. TRANSCRIPTION (RNA SYNTHESIS) 3. TRANSLATION (PROTEIN SYNTHESIS)

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).

The Genetic Dogma : is the flow of genetic material from DNA to DNA, from DNA to RNA and from RNA to protein synthesis.

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 due to mutations or any change in the structure of DNA will eventually cause a Genetic Disease.

DNA Structure
Evidence that DNA is the genetic material:
Previously, it was believed that protein is the genetic material that transfers genetic information from one generation to the next. But later they discovered that protein is not the genetic material, its actually DNA. This discovery was achieved by doing many experiments. One of the most important experiments was DNA Transformation". 1) DNA Transformation experiments: - Objective: to prove that DNA is the carrier of the genetic information. - These experiments have been carried 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 nonvirulent of the same strain. 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 been changed because the genotype has changed). -Results of the experiments:

Since all proteins, carbohydrates and lipids were hydrolyzed by heating; this indicates that DNA is the genetic material. (The genetic material of some viruses is RNA). -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 phenotype into the rough colony's characteristics. 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, and this gene (DNA) will be incorporated into the chromosomal DNA of the egg. When this egg grows and build an organism, this organism will carry the inserted DNA or gene in all or some of its 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 that DNA is the genetic material. - This experiment was carried out by 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).

The experiments that prove DNA as the genetic material

DNA Transformation experiments

Transgenic experiments

The knockout experiments

Structure of DNA:
We are familiar with the structure of DNA from the previous lectures. We talked about the nitrogenous bases (Adenine, Guanine, Thymine, and 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.
Structures of the bases
Purines Pyrimidines

Adenine (A)

Thymine (T)

5-Methylcytosine (5mC)

Guanine (G)

Cytosine (C)

DNA gets modified very frequently in our cells after its 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 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. Promoters: are regions found infront of any gene and they are specific DNA sequences which are responsible for regulating thats gene expression process.

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 .

Since thymine is not foreign to DNA. the repair systm of DNAwill leave 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". Hot islands CG : are the sites where 5mC residues often cluster near the promoters of a gene, and there they cause mutations and thus cancer when they get deaminated . ( CG stands for cytosine and guanine )

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 a glycosidic bond; it's formed between N9 (of the N base) and C1'(of the sugar). -A nucleoside is an N base and a sugar linked by a glycosidic bond.

Nucleoside

[structure of deoxyadenosine]

Nucleotide

-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. We mark the carbons of the sugar with a prime (C1' , C2' ,C3',) to distingush them from the carbons of the N base .

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.

Nomenclature Nucleoside +deoxyribose Nucleotide +phosphate

Base Purines adenine guanine

hypoxanthine

adenosine guanosine
inosine

Pyrimidines thymine cytosine +ribose

thymidine cytidine

uracil

uridine

The structure of the DNA polynucleotide chain

-DNA is composed of two anti-parallel double-stranded polynucleotide chains. -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).

ii). Structure DNA Structure of theof the DNA double helix polynucleotide chain

3 polynucleotide chain 3,5-phosphodiester bond

- 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.

- 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.

A-T base pair

Hydrogen bonding of the bases

G-C base pair

Chargaffs rule: The content of A equals the content of T, and the content of G equals the content of C in double-stranded DNA from any species

According to Chargaffs Rule, the amount of A always equals the amount of T and the amount of G always equals the amount of C. Chargaffs rule was fulfilled when Watson and Crick proposed 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) The resonance phenomenon: is the redistribution of electrons on the surface of the molecule between atoms.

These hydrogen bonds between complementary bases can be changed according to the "resonance phenomena" of these purine and pyrimidine rings, and these changes could cause mutations.

This resonance phenomenon 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 mutations. *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 chains, 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.
Double-stranded DNA
5 3

DNA molecule can exist in many forms (A form, B form, and Z form), but our concern will be DNA in the B form which has many characteristics:

Major groove Minor groove

- Major grooves and minor grooves: B DNA For a gene to be expressed and protein to be synthesized, many proteins (e.g. transcriptional factors) should interact with that gene in order to activate it or sometimes DNA is originally compact when suppress it. Those proteins bind very it's not expressed, when proteins specifically to certain sites on genes, bind to it, DNA will unwrap to be and these sites are called major exposed to all other proteins for grooves". DNA expression . 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 minor grooves are not exposed)
3 5 3

- It has 10 base pairs per turn of the double helix, and the distance between pairs is 3.4 A. *supercoiled DNA: -when a 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) supercoiled DNA

relaxed supercoil
= 10 base pairs / turn

positive supercoil
>10 base pairs / turn

negative supercoil < 10 base pairs / turn

During replication and transcription, positive supercoil will be forming in 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. 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, by using proteins (topoisomerases).

*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 to 3' end or from 3' end to 5' end. These enzymes are important tools in genetic engineering and for constructive purposes such as proofreading during DNA replication.

Chemistry of DNA: *How is the DNA double-helix structure 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 pyrimidines 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 but additive as they are tremendous in number; so they will form a strong force that will stabilize the helical structure.
Stacking interactions

Charge repulsion

Model of double-stranded DNA showing three base pairs

4) Electrostatic interactions: (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 (Histones and Protamines) bind to the negative charges of the phosphate groups and stabilize the double helix structure.
Histones: are small basic proteins, important for expression and stabilizing the double helix because of the positive-negative interaction. Why are they positively charged? Because they are rich with Lys and Arg amino acids which are positively charged at physiological pH.

Charge repulsion

Denaturation of DNA - DNA denaturation is simply the

separation of the two strands by overcoming the forces that stabilize the DNA 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.
Denaturation of DNA
Double-stranded DNA Strand separation and formation of single-stranded random coils

Extremes in pH or A-T rich regions high temperature denature first

Cooperative unwinding of the DNA strands

-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.

Electron micrograph of partially melted DNA

Double-stranded, G-C rich DNA has not yet melted

A-T rich region of DNA has melted into a single-stranded bubble

The regions that have denatured first upon exposure to heat, pH or any other factor, they A-T rich regions melt first, followed by G-C rich regions are rich in A-T base pair, while other regions which are rich in the base pairs G-C will denature later. This difference in the 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.

Hyperchromicity:
*What is Hyperchromicity of DNA? It's a phenomenon 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.
Hyperchromicity

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.
Absorbance maximum for single-stranded DNA

Absorbance

Absorbance maximum for double-stranded DNA

220

260

300

The absorbance at 260 nm of a DNA solution increases when the double helix is melted into single strands.

Hyperchromicity is important in studying the melting curve of DNA (denaturation of DNA by heat) and this will be discussed next lecture.

The End
Done by Rinad Al-Ali