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An important part of our approach to biology is to think concretely about the molecules we are considering. A kilobase (that is, 103 base pairs) of DNA is therefore about 0.34 μm in length. A bacterium, like E. coli, has ~ 3 x 106 base pairs of DNA – that is a DNA molecule almost a millimeter in length, or about 500 times the length of the cell in which it finds itself. To accomplish this, the DNA molecule is associated with specific proteins; the resulting DNA:protein complex is known as chromatin.
The study of how DNA is regulated is the general topic of epigenetics (on top of genetics), while genetics refers to the genetic information itself, the sequence of DNA molecules. If you consider a particular gene (based on our previous discussions) you will realize that to be expressed, transcription factor proteins must be able to find (by diffusion) and bind to specific regions (defined by their sequences) of the DNA in the gene’s regulatory region(s). But the way the DNA is organized into chromatin, particularly in eukaryotic cells, can dramatically influence the ability of transcription factors to interact with and bind to their regulatory sequences. For example, if a gene’s regulatory regions are inaccessible to protein binding because of the structure of the chromatin, the gene will be “off” (unexpressed) even if the transcription factors that would normally turn it on are present and active. As with essentially all biological systems, the interactions between DNA and various proteins can be regulated.
Different types of cells can often have their DNA organized differently through the differential expression and activity of genes involved in opening up (making accessible) or closing down (making inaccessible) regions of DNA. Accessible, transcriptionally active regions of DNA are known as euchromatin while DNA packaged so that the DNA is inaccessible is known as heterochromatin. A particularly dramatic example of this process occurs in female mammals. The X chromosome contains ~1100 genes that play important roles in both males and females278. But the level of gene expression is (generally) influenced by the number of copies of a particular gene. While various mechanisms can compensate for differences in gene copy number, this is not always the case. For example, there are genes in which the mutational inactivation of one of the two copies leads to a distinct phenotype, a situation known as haploinsufficiency. This raises issues for genes located on the X chromosome, since XX organisms have two copies of these genes, while XY organisms have only one279. While one could imagine a mechanism that increased expression of genes on the male’s single X chromosome, the actual mechanism used is to inhibit expression of genes on one of the female’s two X chromosomes. In each XX cell, one of the two X chromosomes is packed into a heterochromatic state, more or less permanently. It is known as a Barr body. The decision as to which X chromosome is packed away (“inactivated”) is made in the early embryo and appears to be stochastic - that means that it is equally likely that in any particular cell, either the X chromosome inherited from the mother or the X chromosome inherited from the father may be inactivated (made heterochromatic). Importantly, once made this choice is inherited, the offspring of a cell will maintain the active/inactivated states of the X chromosomes of its parental cell. Once the inactivation event occurs it is inherited vertically280. The result is that XX females are epigenetic mosaics, they are made of clones of cells in which either one or the other of their X chromosomes have been inactivated. Many epigenetic events can persist through DNA replication and cell division, so these states can be inherited through the soma. A question remains whether epigenetic states can be transmitted through meiosis and into the next generation281. Typically most epigenetic information is reset during the process of embryonic development.
DNA: Structure, Function, Packaging and Properties (With Diagram)
Let us make an in-depth study of the deoxyribonucleic acid. After reading this article you will learn about: 1. Deoxyribonucleic Acid (DNA) 2. Structure of DNA 3. Functions of DNA 4. Packaging of DNA and 5. Physical Properties of DNA.
Deoxyribonucleic Acid (DNA):
Deoxyribonucleic acid, also abbreviated as DNA, is the principal informational macromolecule of the cell, which stores, translates and transfers the genetic information. In the prokaryotes, the DNA is found mostly in the nuclear zone. In eukaryotes it is found in the nucleus, mitochondria and chloroplast. The present understanding of the storage and utilization of the cell’s genetic information is based upon the discovery of the structure of DNA by Watson and Crick in 1953.
Structure of DNA:
Double Helical Structure of DNA (Watson and Crick Model):
The three dimensional structure of DNA as proposed by Watson and Crick and the recent advances in it are summarized here:
1. DNA is made of two helical chains coiled around the same axis, to form a right-handed double helix.
2. The two chains in the helix are anti-parallel to each other, i.e., the 5′-end of one polynucleotide chain and the 3′-end of the other polynucleotide chain is on the same side and close together.
Double helical structure of DNA
3. The distance between each turn is 3.6 nm (formerly 3.4 nm).
4. There are 10.5 nucleotides per turn (formerly 10 nucleotides).
5. The spatial relationship between the two strands creates major and minor grooves between the two strands. In these grooves some proteins interact.
6. The hydrophilic backbones of alternating deoxyribose and negatively charged phosphate groups are on the outside of the double helix.
7. The hydrophobic pyrimidine and purine bases are inside the double helix, which stabilizes the double helix of the DNA.
8. The double helix is also stabilized by inter-chain hydrogen bond formed between a purine and pyrimidine base.
9. A particular purine base, pairs by hydrogen bonds, only with a particular pyrimidine base, i.e., Adenine (A) pairs with Thymine (T) and Guanine (G) pairs with Cytosine (C) only.
10. Two hydrogen bonds pairs Adenine and Thymine (A = T), whereas three hydrogen bonds pairs Guanine and Cytosine (G ≡ C).
11. The base pairs A = T and G ≡ C are known as complementary base pairs.
12. Due to the presence of complementary base pairing, the two chains of the DNA double helix are complementary to each other.
Hence the number of A’ bases are equal to the number of T’ bases (or ‘G’ is equal to ‘C) in a given double stranded DNA.
13. One of the strands in the double helix is known as sense strand, i.e., which codes for RNA/proteins and the other strand is known as antisense strand.
Different Structural Forms of DNA:
The DNA molecules exist in four different structural forms or organizations under different physiological conditions or in different cells or at different points in the same DNA.
Functions of DNA:
The base sequence of the DNA constitutes the informational signal called the genetic material. This nucleotide base sequence enables the DNA to function, store, express and transfer the genetic information. Hence it programs and controls all the activities of an organism directly or indirectly throughout its life cycle.
(a) DNA stores the complete genetic information required to specify (form) the structure of all the proteins and RNA’s of each organism.
(b) DNA is the source of information for the synthesis of all cellular body proteins. Some of the proteins are structural proteins and some are enzymes. These enzymes arrange micro-molecules to form macromolecules. These macromolecules are arranged to form supra-molecular complexes or cell organelles which associate to form cells. These cells group to form tissues which in turn form different organs of a body, specifically peculiar to that organism during foetal develop­ment, growth and repair. Hence DNA programs in time and space the orderly biosynthesis of cells and tissue components.
(c) It determines the activities of an organism throughout its life cycle, i.e., the period of gestation, birth, maturity, senescence and death.
(d) It determines the individuality and identity of a given organism.
(e) It duplicates (replicates to form two daughter DNA) itself and transfers one of the copy to the daughter cell during cell division, thus maintaining the genetic material from generation to generation.
Packaging of DNA:
Packaging of DNA within the Cells:
The length of DNA molecules existing in a particular cell is much longer than the long dimensions of the cell or the organelle where they exist. The contour length (i.e., its helix length) of a double stranded DNA can be calculated from the molecular weight, presuming the average molecular weight of each nucleotide pair to be about 650 Da and there is one nucleotide pair for every 0.36 nm of the duplex.
Accordingly the length of the smallest DNA is 1938.96 nm, belonging to the ɸX174 virus (in duplex form), whose particle long dimension is 25 nm. On the other hand the total contour length of the entire DNA in a single human cell is about 2 metres and the cell nucleus is just 5-10 nm in diameter. These long DNA molecules are very tightly compacted, so as to fit into the cell.
This packing is possible due to DNA getting further coiled into different fashions. The linear double helical DNA called the relaxed DNA, bends or twists upon its own axis, which is called as DNA coiling. This coiled DNA further coils upon itself to form the DNA supercoil, just like the telephone cord wire from the base of the phone to the receiver.
The degree of DNA supercoiling depends upon the type of the cell/organelle to be packed and is coiled in such a manner that DNA can easily be accessible to the enzymes/proteins for all of its functions like replication and transcription.
There are possibilities of two types of DNA supercoiling:
(a) Solenoidal, wherein the DNA coils in a spherical fashion upon itself and
(b) Plectonemic, wherein the DNA coils back upon its reverse length in the form of pleats. Further the supercoiling and packing of DNA differ in the prokaryotes (i.e., those lacking a true nuclear envelope) and in the eukaryotes (i.e., those having a nuclear membrane).
(a) Packaging of viral DNA:
Though the viral DNA is much smaller than a bacterial or eukaryotic DNA, its contour length is much bigger than the long dimensions of the viral particle in which they are found. The DNA of bacteriophage T2 is 3500 times its particle diameter. The long dimensions of different viral particle/the contour length of their DNAs respectively in nanometers is T2-210/65520 Lamdaphage-190/17460 Ti-78/14376 and φX174 (in duplex form)-25/1938. In order to get themselves packed within the particle, most of these viral DNAs are linked covalently by the ends and, therefore, form an endless belt and thus become circular. Some of the single stranded DNA (φX174) becomes double stranded and circular.
(b) Packaging of bacterial DNA:
The length of E.coli cell is 2 micro-meters and its complete DNA molecule (the complete DNA molecule of an organism is called the chromosome) is 1.7 mm long, which exists as a single, covalently closed double stranded circular molecule, coiled and packed within nucleon of the cell.
This circular chromosome is organized in a scaffold-like structure, which folds the chromosome into looped domains. These domains are further coiled around some basic proteins, called Hu proteins (Mw = 10 000). In addition to the nucleosomal DNA the bacteria contains some small circular supercoiled non-chromosomal DNA-called plasmids.
(c) Organization and packing of eukaryotic DNA:
All the chromosomal DNA of an eukaryotic cell is embedded in a membranous cellular organelle called the nucleus. The eukaryotic DNA, in the nucleus is linear and not circular. In the non-dividing resting cell all the DNA of the cell forms a fine filamentous network in the nucleus called the chromatin. During cell division the chromatin network is subdivided into defined number and shaped chromosomes, their diploid number (pairs) depends upon the species of organism.
The normal chromosome num­ber in humans is 46 (23 pairs). Each chromosome has a central axis called the centromere, from which two arms of DNA project out and each is referred to as chromatid. Each chromosome differs in size and shape within a given organism.
The 2 metre long eukaryotic human cell DNA is to be packed in the cell of about 5-10 micrometer in diameter. In order to facilitate its package, the helical DNA molecule is bound, tightly around beads of basic proteins called histones, which are spaced at regular intervals. The complexes of histones and DNA are called nucleosomes. Each nucleosome contains eight histone molecules, two copies each of H2A, H2B, H3 and H4 winding 146 base pairs of DNA.
Between the two nucleosomes there is a spacer DNA of 54 base pairs with a single histone molecule (H1). Wrapping DNA around a nucleosome compacts it to about seven-fold. These nucleosomes are organized very close together to form a structure, simply called 30 nm fibre. This provides 100-fold compaction of DNA. The 30 nm fibre then forms plectonemic pleats, called loops.
Six such loops are bounded by scaffold attachment to proteins (histone- H1/topoisomerase-II) to give rise to a cluster of loops called rosette. 30 such rosettes bunch together to form a single coil, 10 such coils (like phone cord) forms a chromatid and the two chromatids are linked together by a highly repetitive base sequence rich in AT base pairs called satellite DNA, which is the centromere. Two chromatids with a centromere form a chromosome.
Physical Properties of DNA:
When DNA is subjected to extremes of pH or temperatures above 80 to 90 degree centigrade, it gets denatured and the double helical structure is unfolded due to disruption of hydrogen bonds between the bases and the hydrophobic interactions of the bases. Finally, the two strands separate completely from each other. This is melting of DNA. The temperature at which a given DNA is denatured to about 50% is known as TM.
Different DNA melts at different temperatures, which depends upon the G ≡ C content of that DNA. Higher the G ≡ C content, higher is the melting temperature (TM) and vice-versa. When the temperature or pH is slowly brought back to normal biological range, the two strands will automatically rewind or anneal and will again form the same double helical structure. If the temperature is suddenly cooled down, then the two strands remain separated and exist as single strands.
(b) Buoyant Density:
When DNA is centrifuged at high speeds in a concentrated solution of caesium chloride-(CsCI), the CsCl will form a density gradient (ascending) and the DNA will remain stationary or buoyant at a point in the tube where its density is equal to the density of CsCI at that point. Different DNA will have different densities, which again depend upon the GsC content of that DNA. Higher the G = C content, higher is the buoyant density of that DNA and vice versa.
Measurement of these two characters, viz., melting temperature and buoyant density will enable us to calculate the proportions of G ≡ C and A = T pairs in that DNA, which indirectly helps in deducting the gene sequence.
The Structure and Function of Chromatin
Chromatin is a complex of macromolecules composed of DNA, RNA, and protein, which is found inside the nucleus of eukaryotic cells. Chromatin exists in two forms: heterochromatin (condensed) and euchromatin (extended). The primary protein components of chromatin are histones that help to organize DNA into “bead-like” structures called nucleosomes by providing a base on which the DNA can be wrapped around. A nucleosome consists of 147 base pairs of DNA that is wrapped around a set of 8 histones called an octomer. The nucleosome can be further folded to produce the chromatin fiber. Chromatin fibers are coiled and condensed to form chromosomes. Chromatin makes it possible for a number of cell processes to occur including DNA replication, transcription, DNA repair, genetic recombination, and cell division.
Chromatin, Chromosomes and Chromatids
People often confuse these three terms: chromatin, chromosome, and chromatid. While all of those three structures are composed of DNA and proteins within the nucleus, each is uniquely defined.
As mentioned above, chromatin is composed of DNA and histones that are packaged into thin, stringy fibers. The chromatin undergoes further condensation to form the chromosome. So the chromatin is a lower order of DNA organization, while chromosomes are the higher order of DNA organization.
Chromosomes are single-stranded groupings of condensed chromatin. During the cell division processes of mitosis and meiosis, chromosomes replicate to ensure that each new daughter cell receives the correct number of chromosomes. A duplicated chromosome is double-stranded and has the familiar X shape. The two strands are identical and connected at a central region called the centromere.
A chromatid is either of the two strands of a replicated chromosome. Chromatids connected by a centromere are called sister chromatids. At the end of cell division, sister chromatids separate and become daughter chromosomes in the newly formed daughter cells.
The Function of Chromatin
This is the most fundamental function of chromatin: compactification of long DNA strands.The length of DNA in the nucleus is far greater than the size of the compartment in which it is stored. To fit into this compartment the DNA has to be condensed in some manner. Packing ratio is used to describe the degree to which DNA is condensed. To achieve the overall packing ratio, DNA is not packaged directly into structure of chromatin. Instead, it contains several hierarchies of organization.
The first level of packing is achieved by the winding of DNA around the nucleosome, which gives a packing ratio of about 6. This structure is invariant in both the euchromatin and heterochromatin of all chromosomes. The second level of packing is the wrapping of beads in a 30 nm fiber that is found in both interphase chromatin and mitotic chromosomes. This structure increases the packing ratio to about 40. The final packaging occurs when the fiber is organized in loops, scaffolds and domains that give a final packing ratio of about 1,000 in interphase chromatin and about 10,000 in mitotic chromosomes.
Transcription is a process in which the genetic information stored in DNA is read by proteins and then transcribed into RNA, and the RNA will later be translated into functional proteins. If the chromatin gets strengthened and restricts access to the read proteins, there are no transcription occurs. Euchromatin, an extended type of chromatin, can conduct the process of transcription. While heterochromatin, the condensed type of chromatin, is packed too tightly for DNA to be read by proteins.
Fluctuations between open and closed chromatin may contribute to the discontinuity of transcription, or transcriptional bursting. Other factors may probably be involved, such as the association and dissociation of transcription factor complexes with chromatin. The phenomenon, as opposed to simple probabilistic models of transcription, can account for the high variability in gene expression occurring between cells in isogenic population
Chromatin and DNA Repair
The packaging of DNA into the chromatin presents a barrier to all DNA-based processes. Due to the high dynamic arrangement of proteins and DNA, chromatin can readily change its shape and structure. Chromatin relaxation occurs rapidly at the site of a DNA damage, which allows the repair proteins to bind to DNA and repair it.
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