1. Introduction
Genes that are located on chromosomes contain information which, when passed to a new generation, influences the form and characteristics of the offspring. Until 1944, it was not clear what chemical component of the chromosomes makes up genes and constitutes the genetic material. Because chromosomes were known to have both a nucleic acid and a protein component, both were possible candidates.
For a molecule to serve as the genetic material, it must possess 4 major characteristics as follows.
Self replication with absolute fidelity. The molecule responsible for genetic information should have the capability to act as a template for an accurate copy of itself during replication so that progeny cells have the same genetic information as the parent cell. Once the genetic material is doubled in amount, it must then be partitioned equally into 2 daughter cells. During he formation of gametes, the genetic information is also replicated but is partitioned so that each cell gets only half of the original amount of genetic material.
Stable storage of genetic information. The genetic material must have a high capacity for storage of genetic information for an organism’s cell structure, function , development and reproduction. The hereditary material should be stable during the life of a cell; otherwise, information might be gained, lost or altered. It must not change easily due to age, nutrition and environment.
Expression of genetic information. The chemical composition of the genetic material must allow the molecule to code information for many different characteristics.
Variation by mutations. The hereditary material should be subject to small changes or mutations. Without variation, organisms would be incapable of change and adaptation and evolution will not occur.
2. Evidence that DNA is the Hereditary Material
The metabolic stability of DNA. The consistency of DNA in the cell. The distribution of DNA. The correlation between mutagens and their effect on DNA. In mutagenesis. Experiments on bacterial transformation. Experiments to identify the transforming principle. Hershey and Chase bacteriophage experiments.
3. Chemical Composition of DNA and RNA
There are 2 types of nucleic acids namely deoxyribonucleic acid and ribonucleic acid. They are called nucleic acids because they can be isolated from the nuclei and are acidic.
Both DNA and RNA are macromolecules. They have a molecular weight of at least a few thousand Daltons. DNA and RNA are polymers made up of 4 different monomeric units called nucleotides. Each nucleotide consists of a pentose sugar, a nitrogenous base and a phosphate group.
3.1 Pentose Sugar
For RNA, the pentose sugar is ribose and for DNA, the sugar is deoxyribose. The 2 sugars differ by the chemical; groups attached to the 2nd carbon; a hydroxyl group in ribose and a hydrogen atom in deoxyribose.
3.2 Nitrogenous Base
The nitrogenous bases fall into 2 classes: the purines and the pyrimidines. The purines are 9-membered 2 ringed bases. They are adenine (A) and guanine (G). Both are found in DNA and RNA. Pyrimidines are 6-membered single ringed bases. There are 3 types: cytosine (C) found both in DNA and RNA; thymine (T) found only in DNA and uracil (U) found only in RNA.
Because DNA contains deoxyribose sugar, and the pyrimidine thymine, while RNA contains ribose sugar and the pyrimidine uracil, these 2 nucleic acids have different chemical and biological properties. The presence of the 2’OH on the ribose sugar enables RNA to be degraded with alkali while DNA is resistant to the treatment. The cellular enzymes that catalyze nucleic acid syntheses and degradation are usually DNA or RNA specific.
3.3 Nucleosides
A nucleoside is a compound that consists of a base and a sugar covalently linked together. It differs from a nucleotide by lacking a phosphate group in its structure. In a nucleoside, a base forms a glyosidic linkage with the sugar. The glycosidic linkage is from the 1’ carbon of the sugar to he 9 nitrogen atom of the purine base or the 1 nitrogen atom of the pyrimidine base. This reaction occurs with the elimination of water and therefore is a condensation reaction. When the sugar is a ribose, the resulting compound is a ribonucleoside, when the sugar is a deoxyribose, the resulting compound is a deoxyribonucleoside.
3.4 Nucleotides
If a phosphate group is added to the nucleoside, it is now called a nucleotide. Phosphoric acid is most commonly esterified to the 5’ carbon of both DNA and RNA with the elimination of water. This linkage is the phosphoester bond. The resulting compounds are called ribonucleotides or dioxyribonucleoides.
3.5 Polynucleotide Chain
To form a polynucleotide chain of DNA and RNA, condensation between 2 adjacent nucleotides occurs. The nucleotides are linked together by a covalent bond between the phosphate group of one nucleotide and the hydroxyl group on the 3’ carbon of the pentose sugar of the other nucleotide.
The linkage between the 2 nucleotides involves a phosphate group linked to 2 sugars. This is known as the phosphodiester bond, since the phosphate group is linked to the hydroxyl groups on the 2 sugars by means of 2 ester bonds on both sides. Each bond has a 3’ carbon and a 5’ carbon end.
2 joined nucleotides form a dinucleotide, and 3 forms a trinucleotide and so forth. Short chains consisting of approximately 20 nucleotides are called oligonucleotides. Longer chains are called polynucleotides.
The 2 ends of the polynucleotide chain are not the same. The chain has a 5’ carbon at one end and a 3’ carbon at the other end. This asymmetry is referred to as the polarity of the chain.
4. Levels of Structure in Nucleic Acids
The primary structure is the order of bases in the polynucleotide sequence. The secondary structure is the 3D conformation of the backbone (double helix). The tertiary structure is specifically the super-coiling of the molecule. The quaternary structure is the interactions with proteins with the nucleic acids.
5. Physical Structure of DNA
Most DNA consists of 2 polynucleotide strands wound around together in a right-handed helix.
5.1 Composition of DNA
The DNA was known to be composed of bases and sugars and phosphate groups linked together as a polynucleotide chain.
5.2 Base Composition Studies
By chemical treatment, Erwin Chargaff hydrolyzed the DNA and quantified the purines and pyrimidines released.
In all the DNAs, the amount of the purines was equal to the amount of the pyrimidines. The amount of A was equal to that of T and the amount of G was equal to that of C. In comparisons of DNAs from different organisms, the A/T and te G/C ratios were always the same. However the (A+T)/(G+C) ratio varied widely in DNAs of different species.
5.3 X-ray Diffraction Analysis
When fibers of DNA molecules are subjected to a beam of parallel x-rays, these rays are diffracted by the atoms of the DNA molecules in specific patterns according to the atomic weight, spatial arrangement of the molecules and molecule’s atomic structure. These rays are recorded on a photographic plate.
By analyzing the photograph, information of the DNA’s structure can me obtained. The diffraction patterns indicated that the DNA molecule is organized in a highly ordered helical structure. A periodicity of 3.4 angstroms was detected within the structure of the molecule which suggested that the bases were stacked like coins on top of one another.
5.4 Watson-Crick Model
DNA molecule consists of 2 polynucleotide chains coiled around a central axis, forming a right-handed double helix. The 2 strands wind around each other in a clockwise fashion.
The diameter of the helix is 2 nm
The 2 chains are anti-parallel. That is, the 2 strands are orientated in opposite directions with on strand orientated in the 5’ to 3’ way and the other 3’ to 5’.
The sugar-phosphate backbones are on the outsides of the double helix while the bases are orientated toward the central axis. The bases of both chains are flat structures, orientated perpendicularly to the long axis of the DNA. They are stacked on top one another and are located on the inside of the structure.
The nitrogenous bases of opposite strands are bonded together by relatively weak hydrogen bonds. Only specific pairings observed are A with T (2 hydrogen bonds) and G with C 93 hydrogen bonds). The specific pairs are called complementary base pairs, so the nucleotide sequence in one strand dictates the sequence on the other strand.
The base pairs are 0.34 nm apart in the DNA helix. A complete turn of the helix is 3.4 nm long; there are 10 base pairs per turn. Each base pair is twisted 36 degrees clockwise with respect to the previous base pair.
Because of the way the bases bond with each other, the 2 sugar-phosphate backbones of the double helix are not equally spaced along the helical axis. This result in grooves of unequal size between the backbones called the major groove and the minor groove. Both of these grooves are large enough to allow protein molecules to make contact with the bases.
5.5 Base-Pairing in DNA
The anti-parallel nature of the 2 chains is a key part of the double-helix model. Given the constraints of the bond angles of the various nucleotide components, the double-helix could not be constructed easily if both chains ran parallel to one another.
The most important aspect of the DNA double helix is the specificity of base pairing. A must pair with T and C must pair with G because the steric and hydrogen bond factors. The 2 strands of DNA double helix are thus said to be complementary. Complimentarity is important in DNA replication and gene expression.
5.5.1 Steric Factors
According to the X-ray data, the double helix has a uniform diameter of 2 nm. A and G are purines, nitrogenous bases with 2 organic rings. C and T are pyrimidines which have a single ring. Thus, purines are about twice as wide as pyrimidines. A purine-purine pair is too wide and a pyrimidine-pyrimidine pair is too narrow to account for the 2 nm diameter of the double helix. The solution is to always pair a purine with a pyrimidine.
5.5.2 Hydrogen Bond Factors
A and T always have 2 hydrogen bonds and C and G always have 3 hydrogen bonds with each other.
5.6 Factors Contributing to the Stability of DNA
5.6.1 Hydrogen Bonds
A hydrogen bond is a weak electrostatic attraction between a covalently bonded hydrogen atom and an atom with an unshared electron pair.
The hydrogen atom assumes a partial positive charge, while the unshared pair assumes a partial negative charge. These opposite charges are responsible for the weak chemical attractions.
Although 2 or 3 hydrogen bonds taken alone are energetically weak, 2000-3000 bonds in tandem, which would be found in 2 long polynucleotide chains, provide great stability to the helix.
Hydrogen bonding between 2 strands can be reversibly disrupted by heat. Is a sample of DNA is heated in boiling water; single strands of DNA are formed. The separation of DNA into single-stranded molecules is referred to as denaturation. Slow cooling of denatured DNA can result in the reformation of the double stranded DNA as the complementary strands eventually find each other and this is referred to as renaturation.
5.6.2 Stacking Forces
A non-covalent force exists between adjacent bases stacked one upon another. The atoms in one pair of bases are in close contact with those of the adjacent base pairs. They are held together by van der Waals forces or stacking forces. Cumulatively, these forces are very important in maintaining the double helix of the DNA.
5.6.3 Arrangement of Sugars and Bases along the Axis
The hydrophobic nitrogenous bases are stacked almost horizontally on the interior of the axis and are thus shielded from water. The hydrophilic sugar-phosphate backbone is on the outside of the axis, where both components can interact with water. These molecular arrangements provide significant chemical stabilization to the helix. Moreover, all potentially reactive side groups of the deoxyribose sugar and the nitrogenous bases are already involved in the formation of bonds.
5.6.4 Redundancy of Genetic Information
Genetic information that is ultimately translated into proteins lies in the specific order of base pairs. The information is redundant in the DNA molecule in the sense that the specific order of bases on one chain determines the specific order of bases on the other chain. This redundancy helps to maintain the integrity of genetic information. If one strand is altered, the cell discards the damaged strand and makes a new accurate one by using the remaining intact strand as the template, following the complementary base-pairing rules.
5.6.5 Organization of DNA in Eukaryotic Chromosomes
Eukaryotic DNA is wound around histones in a structure called the nucleosome. Histones are proteins that are rich in basic (positively charged) side chains. The positive charges can form ionic bonds with the negatively charged phosphate groups of DNA thus stabilizing it.
5.6.6 Formation of Hairpin Loops in Single Stranded DNA
Naturally occurring single stranded DNA also exists in some phages and viruses. Because the stability of double stranded DNA is much greater, single stranded forms of DNA tend to fold back upon themselves whenever possible to form double stranded helices with loops. These anti-parallel duplex structures are called hairpins with a stem (paired bases) and a loop (unpaired bases)
6. DNA Replication
Living organisms perpetuate their kind through reproduction which entails the faithful transmission of genetic information of the parents to their progeny. Since genetic information is stored in DNA, the replication of DNA is central to all of biology.
6.1 Models of DNA Replication
One of the most significant features of the double helical model of DNA is that it immediately suggests a mechanism for DNA replication. There is a paper published suggestion how DNA might duplicate itself.
Now our model of DNA is a pair of templates, each of which is complementary to each other. We imagine that prior to duplication, the hydrogen bonds are broken, and the 2 chains unwind and separate. Each chain then acts as a template for the formation onto itself of a new companion strand, so that eventually we have 2 pairs of chains, where we only had one before. The sequence of the base pairs will have been duplicated exactly.”
The 2 DNA strands are complementary; each stores the information necessary to reconstruct the other. As the DNA double helix is progressively unwound from one end, each parental strand directs the synthesis of a new complementary strand according to the specific base-pairing requirements; each parental strand serves as a template for a new complementary strand. When replication is completed, there would be 2 progeny DNA double helices, each consisting of one parental DNA strand and one new DNA strand. Each progeny double helix is an exact replica of the original molecule. This is called the semi-conservative model since each progeny retains one of the parental strands.
In the conservative model, the 2 parental strands of DNA remain together or reanneal after replication and as a whole serve as a template for the synthesis of new progeny DNA double helices. Thus one of the 2 progeny DNA molecules is actually he parental double-stranded DNA molecule and the other consists of totally new material.
In the dispersive model, the parental double helix is cleaved into double stranded DNA segments templates which acts as templates for the synthesis of new double stranded DNA segments. These segments reassemble into complete DNA double helices, with parental and progeny DNA segments interspersed. Thus, the parental DNA has actually become dispersed throughout both progeny molecules.
6.2 Process of DNA Replication
DNA replication has been shown to be a complex process requiring a number of enzymes and other proteins and even the participation of RNA.
6.2.1 General Features of DNA Replication
The initiation of DNA replication requires the local separation of the 2 DNA strands at a specific DNA sequence called an origin of replication.
2 replication forks are formed at the origin and the replication proceeds simultaneously in both directions. The Y-shaped forks are at the end of a replication bubble that grows in size as replication continues bidirectionally. In contrast to the circular bacterial chromosome, each linear eukaryotic chromosome has hundreds or thousands of replication origins. Multiple replication bubbles form and eventually fuse, thus speeding up the copying of very long DNA molecules.
The individual unit of replication is called the replicon. A replicon is a self-replicating segment of a chromosome that includes an origin from which replication proceeds bidirectionally.
6.2.2 Molecular Aspects of DNA Replication in Prokaryotes
Reference is made to E.coli as DNA replication is best understood in this prokaryote. The detailed mechanism of DNA replication involves the following steps.
6.2.2.1 Initiation of DNA Replication at the Origin of Replication
DNA replication usually starts at a specific site on the chromosome called the origin of replication. An initiator protein binds to this sequence. Using energy from ATP, the initiator protein slightly unwinds the double helix, forming a replication bubble and allowing the rest of the DNA replication machinery access to the single-stranded DNA.
6.2.2.2 Unwinding and Separation of DNA strands
For DNA replication to proceed, the DNA must unwind to expose the single strands to the enzymes responsible for copying them. This aspect of DNA replication is known to involve at least 3 kinds of proteins with distinct functions.
The proteins directly responsible for unwinding the DNA double helix are called helicases. These enzymes use the energy of ATP to unwind the DNA in advance of the replication fork, breaking the hydrogen bonds as they go.
The unwinding would create an intolerable amount of super-coiling and possibly tangling in the rest of the DNA were it not for the actions of DNA topoisomerases. These enzymes can form swivels in the DNA by making and then quickly resealing single or double stranded breaks in he double helix.
Of the 10 or so topoisomerases found in E.coli, the one that is the most important is DNA gyrase that cuts both DNA strands. Using energy from ATP, gyrase relaxes super-coiling and prevents over-winding of DNA ahead of the replication fork. Gyrase has special roles in both the initiation and completion of replication in E.coli-in opening up the double helix at the origin of replication and in separating the linked circles of daughter DNA at the end.
Once strand separation has begun, molecules of single-strand binding protein (SSB) quickly attach to the exposed single strands at the replication fork in such a way that they do not cover the nitrogenous bases. The SSB proteins stabilize the single stranded DNA to which they bind, to prevent the reformation of the double helix or the formation of hydrogen bonds between bases at different parts of the same strand.
6.2.2.3 Priming Synthesis of new DNA Strand
A Short piece of RNA (3-10 nucleotides) is synthesized first an serves as a primer for DNA synthesis. This is an oligonucleotide onto which deoxyribonucleotides are added. These RNA primers are synthesized by a DNA dependant RNA polymerase called Primase. Primase is a specific kind of RNA polymerase that is involved only in the process of DNA replication. Like all other RNA polymerases, primases can initiate a new polynucleotide strand complementary to the template strand; they do not themselves require a primer.
6.2.2.4 Elongation of new DNA Strands
Enzymes capable of adding successive nucleotides to a growing DNA strand are called DNA polymerases. No known DNA polymerase can initiate the synthesis of a DNA strand; they can only catalyze the addition of deoxyribonucleotides to a preexisting strand. Thus primase is required in DNA replication.
As nucleotides align with complementary bases along a template strand of DNA, they are added by DNA polymerase, one by one, to the growing end of the new DNA strand. The substrates for DNA polymerase are deoxyribonucleoside triphosphates. DNA polymerase catalyzes the covalent bonding of nucleotides to the 3’ hydroxyl end of the growing polynucleotide chain.
Each successive nucleotide is linked to the growing chain by a phosphoester bond between the phosphate group on its 5’ carbon and the hydroxyl group on the 3’ carbon of the nucleotide added in the pervious step. Chain elongation therefore always occurs at the 3’ end of the DNA strand and the strand grows in the 5’ to 3’ direction.
6.2.2.4.1 Discontinuous Synthesis
When it became clear that DNA replication always involves the addition of nucleotides to the 3’ end of each growing nucleotide chain, a conceptual problem was evident.
Given the anti-parallel orientation of the 2 strands in a DNA molecule, continuous nucleotide addition along both strands of a replication fork would require synthesis in the 5’ to 3’ direction on one strand and in the 3’ to 5’ direction on the other strand. But all known DNA polymerases function only in the 5’ to 3’ direction.
This was resolved when it was realized that synthesis at each replication fork is continuous in the fork movement for one strand but discontinuous in the direction away from the fork for the other strand. The 2 daughter strands can therefore be distinguished based on their mode of growth.
6.2.2.4.2 Leading Strand
The leading strand is the strand to which nucleotides can be added continuously because it s growing in the 5’ to 3’ direction towards the replication fork as it advances.
6.2.2.4.3 Lagging Strand
The lagging strand grows by synthesis of short pieces called Okazaki fragments in the 5’ to 3’diraction away from the replication fork. Each fragment grows until it meets the adjacent fragment. Okazaki fragments are eventually joined together by DNA ligase, which catalyses the formation of phosphoester bonds between the nucleotides at the 3’ and 5’ ends of adjacent fragments.
Both leading and lagging strands require RNA primers to initiate the synthesis of new DNA strands, although for the leading strand, only one primer is needed at origin of replication.
The removal of RNA primers occurs nucleotide by nucleotide by the 5’à3’ exonuclease activity of DNA polymerase. When DNA polymerase has completed the displacement of RNA primers with DNA nucleotides, DNA ligase joins the DNA fragments together into one continuous strand.
7. DNA Proofreading and Repair
The faithfulness with which DNA sequences are maintained from one generation of cells to the next requires that DNA be replicated very accurately and provision be made for repairing DNA damage that can arise both spontaneously and from exposure to environmental agents. Enzymes proofread DNA during its replication nd repair damage in existing DNA.
7.1 Proofreading during DNA Replication
About 1 in every 100,000 nucleotides incorporated during DNA replication is incorrectly base-paired with the template DNA strand. Such mistakes are corrected by a proofreading mechanism. Proofreading uses the same DNA polymerase that catalyzes DNA synthesis.
In addition to its 5’ à 3’ polymerizing ability, DNA polymerase exhibits 3’ à 5’ exonuclease activity. An exonuclease is an enzyme that degrades DNA from one end. Hence the 3’ à 5’ exonuclease activity of DNA polymerase allows it to remove improperly base-paired nucleotides from the 3’ end of the growing chain. This ability to remove incorrect nucleotides can improve the fidelity of DNA replication to an average of only a few errors for every billion base pairs.
7.2 DNA Damage and Repair
The net rate at which organisms accumulate mutations is low; an average gene retains only 1 mutation every 200,000 years. The underlying rate of DNA mutation is far greater than this number suggests because most damage is repaired shortly after it occurs.
7.2.1 Spontaneous DNA Damage
During DNA replication, spontaneous hydrolysis reactions caused by random interactions of DNA and water molecules around it may lead to depurination (loss of a purine base) or deamination (removal of a base’s amino group). If a DNA strand with missing purines or deaminated bases is not repaired, an erroneous base sequence may be propagated when the strand serves as a template in the next round of DNA replication.
7.2.2 DNA Damage in response to Mutagens
Mutagens are mutation-causing agents in our environment. Environmental mutagens fall into 2 categories: chemicals (base analogues, base-modifying agents, intercalating agents) and radiation (UV radiation, radioactive emissions, X-rays). Sun light is a string source of UV radiation which alters DNA by triggering pyrimidine dimer formation; that is, the formation of covalent bonds between adjacent thymines. This causes a bulge in the DNA double helix and replication is blocked.
7.2.3 Excision Repair
Excision repair corrects mutations that involve abnormal nucleotides using a basic 3 step process. The defective nucleotides are cut out from one strand of the DNA double helix. This step is carried out by repair endonucleases. The missing nucleotides are replaced with the correct ones by DNA polymerases. DNA ligase seals the remaining nick in the repaired strand by forming the missing phosphoester bond.
One function of DNA repair enzymes in our skin cells is to repair genetic damage caused by UV rays of sunlight. The disorder xerodema pigmentosm is caused by an inherited defect in an excision repair enzyme. Individuals with this disorder are hypersensitive to sunlight; mutations in their skin cells caused by UV light are left uncorrected and can lead to skin cancer.
7.2.4 Mismatch Repair
Mismatch repair corrects mutations that involve non-complementary base pairs. It targets errors made during DNA replication which have sometimes escaped normal proofreading mechanisms. A repair endonuclease introduces a single nick in 1 strand and an exonuclease removes a section of DNA, including the mismatch, from the nicked strand. DNA polymerase and ligase then repair the gap, producing the correct base pair.
