Molecular Biology Training Package PDF

Ministry of Higher Education and Scientific Research Northern Technical University College of Health and Medical Techniques/Kirkuk Medical Laboratory Techniques Department Training package In Molecular Biology For 2nd Level Students By Prof. Dr. Asal Az...

Ministry of Higher Education and Scientific Research Northern Technical University College of Health and Medical Techniques/Kirkuk Medical Laboratory Techniques Department Training package In Molecular Biology For 2nd Level Students By Prof. Dr. Asal Aziz Tawfeeq Ph.D. Biotechnology 2023-2024 1|Prof.Dr. Asal Aziz  Molecular Biology Training package for 2nd Level Students// Courses Objective: MB Molecular biology is a science that is concerned with the study of biology at the molecular level, so it overlaps with both microbiology and chemistry in several branches and intersects with biochemistry and genetics in several areas and disciplines. Molecular biology is concerned with the study of the various interrelationships between all Cellular systems, especially the relationships between deoxyribonucleic acid (DNA) and RNA (ribonucleic acid) and the process of protein synthesis. In addition to the mechanisms regulating this process and all vital processes in the body. At the end of this course, the student will be able to understand the three- dimensional structures and structural formations of nucleic acids in humans as well as understanding the molecular basis of the process of transcription, translation, DNA damage and mutations. 2|Prof.Dr. Asal Aziz Lecture No.1: Title of the lecture: Introduction to Molecular Biology &Nucleic acids -What is Molecular Biology? Molecular biology, is the study of chemical and physical structure of biological macromolecules of the cell. It is a branch of biology that is focused especially on nucleic acids (e.g., DNA and RNA) and proteins—macromolecules that are essential to life processes—and how these molecules interact and behave within cells. Molecular biology was first described as an approach focused on the foundations of biological phenomena—uncovering the structures of biological molecules as well as their interactions, and how these interactions explain observations of classical biology. -What is molecular biology used for? The term molecular biology was first used in 1945 by the physicist William Astbury where the field included techniques which enable scientists to learn about molecular processes and were used to efficiently target new drugs, diagnose disease, and better treatment choices. Whereas, recently, molecular biology techniques play a vital role in advancing our understanding of genetics, cell biology, and biochemistry. Their research is used in a variety of fields, including: biotechnology, medicine, agriculture, and environmental science. -Macromolecules examples studied in molecular biology: There are two major classes of biological macromolecules (nucleic acids and proteins), and each is an important component of the cell and performs a wide array of functions. 3|Prof.Dr. Asal Aziz Nucleic acids Nucleic acids and proteins are the basic macromolecules of living organisms. The linkage between nucleic acids and proteins is very close and each macromolecule has its unique function in the cell. - The Function of the Nucleic Acids However, Since Gregor Mendel, the Austrian monk had discovered the basic principles of heredity through experiments in his garden where his observations became the foundation of modern genetics; scientists started to inquire about the “material” which was responsible about the transmission of the traits from one generation to the following. Three macromolecules were questioned as the carrier of the genetic traits they were namely (DNA, RNA and protein). However, after many experiments by many scientists all over the world, in 1952 an experiment was conducted by Hershey and Chase involving the bacteriophage T2 whose DNA was labeled by a radionuclide 32P, and the protein part by a 35S radionuclide. During the infection by a bacteriophage, only the DNA part of the virus entered the cell. This supported the evidence, that DNA was the carrier of the genetic information. However, certain viruses use the ribonucleic acid-RNA) as the carrier of the genetic material. First and foremost, the genetic material must be capable of storing large amounts of information and instructions for all the traits and functions of an organism. This information must have the capacity to vary, because different species and even individual members of a species differ in their genetic makeup. At the same time, the genetic material must be stable, because most alterations to the genetic instructions (mutations) are likely to be detrimental if turned around, errors in the genetic code lead to the synthesis of defective proteins, or to stop the synthesis of proteins overall. 4|Prof.Dr. Asal Aziz -Primary structure of nucleic acid: Nucleic acids were discovered as “nuclein” by a Swiss physician and biologist Miescher in the year 1869, and their name was created by Altmann (1889). For the synthesis of nucleic acids nucleosides are used, which are made of a nitrogenous base and pentose, but the structure of DNA differs than RNA. 2- Deoxyribonucleic acid (DNA) from the beginning DNA was discovered as a major chemical of the nucleus able to transmit large amounts of hereditary information from generation to generation. Although DNA was known to be a very large molecule, it is relatively simple in structure, having an elegant and beautiful incomparable structure in comparison with the other large molecules in the body. It is useful to consider the structure of DNA at three levels of increasing complexity, known as the primary, secondary, and tertiary structures of DNA. ►Primary Structure of DNA The primary structure of DNA consists of a string of nucleotides. (see figure 1.1). Figure (1.1): The composition of a nucleotide in DNA. Each nucleotide is composed of three parts: 1- Phosphate group 2- Sugar (pentose) 3- Nitrogenous base The first component of a nucleotide is the phosphate group, which consists of a phosphorus atom bonded to four oxygen atoms. Phosphate groups are found in every nucleotide and 5|Prof.Dr. Asal Aziz frequently carry a negative charge, which makes DNA acidic. The phosphate is always bonded to the 5-carbon atom of the sugar in a nucleotide. The second component is the sugars of the nucleic acids which is called "pentose sugar". This sugar consists of five carbon atoms, numbered 1´-, 2´-, 3´-, 4´- and 5´- see figure (1.2). In ribose sugar, five carbon atoms are joined by either a hydrogen (-H) or a hydroxyl (-OH) atoms to form a five-sided ring; the fifth (5´-) carbon atom projects upward from the ring. While, in the 2-Deoxyribose sugar, the carbon atom number two is attached to hydrogen atom (-H) instead of a hydroxyl groups (-OH) found attached to the carbon atom number two in ribose sugar making it deficient in an oxygen atom and gaining the name 2´- Deoxyribose. Figure (1.2): The composition of pentose sugars in the nucleic acids. This minor chemical difference in the ribose sugar between DNA and RNA is recognized by all the cellular enzymes that interact with DNA or RNA, thus yielding specific functions for each nucleic acid. Furthermore, the additional oxygen atom in the RNA nucleotide makes it more reactive and less chemically stable than DNA. For this reason, DNA is better suited to serve as the long-term storehouse of genetic information. 6|Prof.Dr. Asal Aziz The third component of a nucleotide is its nitrogenous base, which may be of two types; a purine or a pyrimidine. See figure (1.3). Figure (1.3): The chemical structure of nitrogenous bases in the nucleic acids. Purines are the larger of the two types of nitrogen bases found in DNA and RNA. They are composed of nine atoms that make up the fused rings (5 carbon and 4 nitrogen atoms) that are numbered from one to nine. All ring atoms lie in the same plane. Adenine (A) and Guanine (G) are purines they occur in both DNA and RNA. On the other hand, pyrimidines are composed of six atoms (4 carbon and 2 nitrogen atoms) they are numbered from one to six. Like purines, all pyrimidine ring atoms lie in the same plane and Cytosine (C), Uracil (U) and Thymine (T) are pyrimidines. Each purine consists of a six-sided ring attached to a five-sided ring, whereas each pyrimidine consists of a six-sided ring only. DNA and RNA both contain two purines, adenine and guanine (A and G). There are three pyrimidines found in nucleic acids: cytosine (C), thymine (T), and uracil (U). Cytosine is present in both DNA and RNA; however, thymine is restricted to DNA, and uracil is found only in RNA. In a nucleotide, the nitrogenous base always forms a covalent bond (glycosidic bond/ N-glycosidic linkage) with the carbon number one atom of the sugar. However, a deoxyribose (or ribose) sugar and a base together are referred to as a nucleoside. Figure (1.4): The chemical structure of a nucleoside in the nucleic acid (RNA). 7|Prof.Dr. Asal Aziz On the other hand, a phosphodiester bond is formed between the sugar and the phosphate group in the nucleotide. However, the “backbone” of a DNA molecule in each cell consists of polynucleotide chains composed of many nucleotides connected by covalent bonds, which join the 5´- phosphate group of one nucleotide to the 3´-carbon atom of the ribose sugar in the next nucleotide as shown below in figure (1.5). where those nucleotides were joined together by phosphodiester linkages. Figure (1.5): Polynucleotide chain structure in the nucleic acids. The phosphodiester linkages, are relatively strong covalent bonds; a series of nucleotides linked in this way constitutes a polynucleotide strand. The backbone of the polynucleotide strand is composed of alternating sugars and phosphates; the bases project away from the long axis of the strand and the negative charges of the phosphate groups are frequently neutralized by the association of positive charges on proteins, metals, or other molecules. Thus, the DNA is typically a very long molecule and is therefore, termed a macromolecule, if stretched out straight, would be several centimeters in length. 8|Prof.Dr. Asal Aziz Direction of DNA polynucleotide An important characteristic of the polynucleotide strand is its direction, or polarity. At one end of the strand a phosphate group is attached to the 5´-carbon atom of the sugar in the nucleotide which is therefore, referred to as the 5´- end. On the other hand, a 3´-carbon atom of another sugar molecule is attached to the same phosphate group and referred as the 3´- end. Thus, the polynucleotide chains have a 5'-to- 3' directionality. A fundamental feature of the polynucleotide chain is that its ends are dissimilar. Thus, the 3'- hydroxyl is displayed at one terminus, (or so called the 3'- end), and the 5'- phosphoryl at the other terminus, (or called the 5'- end). The directionality of the polynucleotide chain is very important feature of the DNA since the two ends of the molecule have a very different biochemical properties, and behave very differently in molecular processes. DNA directionality is also important in governing various cellular events. Furthermore, the nitrogenous bases of each polynucleotide chain is bond to each other by hydrogen bonds and align in an antiparallel orientation in the double helix forming the secondary structure of DNA. 9|Prof.Dr. Asal Aziz Lecture No.2: Title of the lecture Secondary Structure of DNA The model of the secondary structure of DNA consists of two polynucleotide strands twisted around each other forming a unique structure called (a double helix). This double helix is composed after the linkage of sugar–phosphate on the outside of the helix, and the staking of the purine and pyrimidine bases bonded by hydrogen bonds in the interior of the molecule forming a complex of two polynucleotide chains twisted around a mutual axis so that, the carbohydrate chain protrude outside the chain and the nucleic acid bases are directed inwards the chain. See figure (2.1). Figure (2.6): Secondary structure of DNA. Still, the basic structure of DNA can be divided into two portions: the external sugar-phosphate backbone, and the internal bases. The sugar phosphate backbone, as its name implies, is the major structural component of the DNA molecule. The backbone is constructed from alternating ribose sugar and phosphate molecules which are highly polar. Because the backbone is polar, it is hydrophilic which means that it likes to be immersed in water. While, the interior portion of a DNA molecule is composed of a series of four 10 | P r o f. D r. A s a l A z i z  nitrogenous bases: adenine (A), guanine (G), thymine (T), and cytosine (C). These bases are non-polar therefore they are hydrophobic (they don't like water). Inside a DNA molecule these bases pair up, A to T and C to G, forming hydrogen bonds that stabilize the DNA molecule. Because the interior bases pair up in this manner, we say that; the DNA double helix is complimentary. It is this sequence of bases inside the DNA double helix that we refer to as the genetic code. However, all of the DNA molecules are built in 5´ to 3´ direction while the other strand lane in the opposite direction making the two strands “antiparallel” showing an opposite chemical polarity. What is the double helix of a DNA? Double helix, as related to genomics, is a term used to describe the physical structure of DNA. A DNA molecule is made up of two linked strands that wind around each other to resemble a twisted ladder in a helix-like shape. Why is DNA double helical? To maximize the efficiency of base-pair packing, the two sugar-phosphate backbones wind around each other to form a double helix, with one complete turn every ten base pairs 11 | P r o f. D r. A s a l A z i z  ►Tertiary Structure of DNA Nucleic acid tertiary structure is the three-dimensional shape of a nucleic acid polymer. RNA and DNA molecules are capable of diverse functions ranging from molecular recognition to catalysis. Such functions require a precise three-dimensional structure. Thus, the DNA tertiary structure is the three-dimensional geometrical formation of the nucleotides and can include B-DNA, A-DNA, and Z- DNA which is determined by the base pair geometry. The most popular forms of DNA A-DNA: It is a right-handed double helix, short and fat compared to B-DNA , occur only in dehydrated cells B-DNA: This is the most common DNA conformation and is a right-handed helix, described by Watson and Crick.... Z-DNA: Z-DNA is a left-handed DNA where the double helix winds to the left in a zig-zag pattern. Long and thin in comparison to B-DNA 12 | P r o f. D r. A s a l A z i z  Types of DNA according to cell location According to its location inside the cell we can divide DNA into more types. The most important is the chromosomal DNA. The mitochondrial DNA and plasmid DNA. 1-Chromosomal DNA Chromosomal DNA can be divided into the certain groups according to function, coding and non-coding regions. The coding DNA determines the sequence of amino in the polypeptide chain (structural genes), or nucleotides order in the RNA types There are more types of non-coding DNA – for example the DNA which has a control and regulatory function (for instance promoters). Some types of DNA have a specific function inside the chromosomes, for instance the repetitive sequences in the region of the centromeres or telomeres. A chromosome is formed from a single, enormously long DNA molecule that contains a linear array of many genes. The human genome contains 3.2 × 109DNA nucleotide pairs, divided between 22 different autosomes and 2 sex chromosomes. Only a small percentage of this DNA codes for proteins or structural and catalytic RNAs. What is the difference between DNA and chromosomal DNA? A chromosome is a long chain of DNA molecules that contains part of all of the genetic material of an organism. DNA is a fundamental molecule that carries the genetic instruction of all living organisms. DNA is packed into chromosomes with the help of special proteins called histones. 13 | P r o f. D r. A s a l A z i z 2-Mitochondrial DNA (mtDNA) In humans, the non-chromosomal DNA is located in the mitochondria. Mitochondrial DNA is the circular chromosome found inside the cellular organelles called mitochondria. Located in the cytoplasm, mitochondria are the site of the cell's energy production and other metabolic functions. Offspring inherit mitochondria — and as a result mitochondrial DNA — from their mother. Mitochondria therefore have their own DNA (mtDNA), circular and double-stranded, closer to a prokaryotic genome than nuclear DNA, with a genetic code slightly different from the universal genetic code found in the nucleus of eukaryotic cells. Mitochondrial DNA (mtDNA) has many special features such as a high copy number in cell, maternal inheritance, and a high mutation rate which have made it attractive to scientists from many fields. Is mitochondrial DNA only found in females? Mitochondrial DNA (mtDNA) is passed from mother to child. Both sons and daughters receive mtDNA, but only daughters pass the mtDNA on to their own children. Since both sons and daughters receive their mother's mtDNA, both men and women can take mtDNA tests. 14 | P r o f. D r. A s a l A z i z What are the differences between mitochondrial and nuclear DNA? Nuclear DNA is located within the nucleus of eukaryote cells and usually has two copies per cell while mitochondrial DNA is located in the mitochondria and contains 100–1,000 copies per cell. Is mitochondrial DNA single or double-stranded? Mitochondrial DNA (mtDNA) is a double-stranded molecule of 16.6 kb How many genes are in mitochondrial DNA? 37 genes This genetic material is known as mitochondrial DNA or mtDNA. In humans, mitochondrial DNA spans about 16,500 DNA building blocks (base pairs), representing a small fraction of the total DNA in cells. Mitochondrial DNA contains 37 genes, all of which are essential for normal mitochondrial function Does mitochondrial DNA have RNA? Thanks to its mtDNA mitochondria possess their own set of tRNAs, rRNAs and mRNAs that encode a subset of the protein subunits of the electron transport chain complexes. 15 | P r o f. D r. A s a l A z i z 3-Plasmids A plasmid is a small circular DNA molecule found in bacteria and some other microscopic organisms. Plasmids are physically separate from chromosomal DNA and replicate independently. Plasmids naturally exist in bacterial cells but, they also occur in some eukaryotes. With no mechanism to penetrate most eukaryotic cells. However, viruses can and are used to transport DNA into eukaryotic cells. Still, the genes carried in plasmids provide bacteria with genetic advantages, such as antibiotic resistance. How plasmids are formed? Plasmids found in nature often give their hosts beneficial traits that allow them to survive in competitive environments. Plasmids derived directly from the environment are sometimes called 'natural' plasmids, to distinguish them from the modified versions we usually work with in the lab. The construction of artificial plasmids is crucial in modern molecular biology. In many cases, plasmids are constructed in vitro by digesting (cutting) DNA fragments with restriction enzymes at specific sites (restriction sites) and then ligating (joining) the resulting fragments. Are plasmids found in eukaryotic mitochondria? Mitochondrial genomes exhibit diverse features among eukaryotes in the aspect of gene content, genome structure, and the mobile genetic elements such as introns and plasmids. 16 | P r o f. D r. A s a l A z i z Lecture No.3: Title of the lecture: Molecular Structure of RNA RNA, abbreviation of ribonucleic acid, a complex compound of a high molecular weight that functions in cellular protein synthesis and replaces DNA (deoxyribonucleic acid) as a carrier of genetic codes in some viruses. RNA consists of ribose nucleotides (nitrogenous bases appended to a ribose sugar) attached by phosphodiester bonds, forming strands of varying lengths. The nitrogenous bases in RNA are adenine, guanine, cytosine, and uracil, which replaces thymine in DNA. The ribose sugar of RNA is a cyclical structure consisting of five carbons and one oxygen. The presence of a chemically reactive hydroxyl (−OH) group attached to the second carbon group in the ribose sugar molecule makes RNA prone to hydrolysis. This chemical lability of RNA, compared with DNA, which does not have a reactive −OH group in the same position on the sugar moiety (deoxyribose), is thought to be one reason why DNA evolved to be the preferred carrier of genetic information in most organisms. The structure of the RNA molecule was described by R.W. Holley in 1965. RNA structure RNA typically is a single-stranded biopolymer. However, the presence of self-complementary sequences in the RNA strand leads to intrachain base-pairing and folding of the ribonucleotide chain into complex structural forms. The three-dimensional structure of RNA is critical to its stability and function, allowing the ribose sugar and the nitrogenous bases to be modified in numerous different ways by cellular enzymes that attach chemical groups (e.g., methyl groups) to the chain. Such modifications enable the formation of chemical bonds between distant regions in the RNA strand, leading to complex contortions in the RNA chain, which further stabilizes the RNA structure. 17 | P r o f. D r. A s a l A z i z Types and functions of RNA Of the many types of RNA, the three most well-known and most commonly studied are messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA), which are present in all organisms. They primarily carry out biochemical reactions, similar to enzymes. Some, however, also have complex regulatory functions in cells. Owing to their involvement in many regulatory processes, to their abundance, and to their diverse functions, RNAs play important roles in both normal cellular processes and diseases. In protein synthesis, mRNA carries genetic codes from the DNA in the nucleus to ribosomes, the sites of protein translation in the cytoplasm. Ribosomes are composed of rRNA and protein. The ribosome protein subunits are encoded by rRNA and are synthesized in the nucleolus. Once fully assembled, they move to the cytoplasm, where, as key regulators of translation, they “read” the code carried by mRNA. A sequence of three nitrogenous bases in mRNA specifies incorporation of a specific amino acid in the sequence that makes up the protein. Molecules of tRNA (sometimes also called soluble, or activator, RNA), which contain fewer than 100 nucleotides, bring the specified amino acids to the ribosomes, where they are linked to form proteins. In addition to mRNA, tRNA, and rRNA, RNAs can be broadly divided into coding (cRNA) and noncoding RNA (ncRNA). 18 | P r o f. D r. A s a l A z i z There are two types of ncRNAs, housekeeping ncRNAs (tRNA and rRNA) and regulatory ncRNAs, which are further classified according to their size. Long ncRNAs (lncRNA) have at least 200 nucleotides, while small ncRNAs have fewer than 200 nucleotides. Small ncRNAs are subdivided into micro RNA (miRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), small- interfering RNA (siRNA), and pi-interacting RNA. The miRNAs are of particular importance. They are about 22 nucleotides long and function in gene regulation in most eukaryotes. They can inhibit (silence) gene expression by binding to target mRNA and inhibiting translation, thereby preventing functional proteins from being produced. Many miRNAs play significant roles in cancer and other diseases. For example, tumor suppressor and oncogenic (cancer-initiating) miRNAs can regulate unique target genes, leading to tumorigenesis and tumor progression. Also, of functional significance are the piRNAs, which are about 26 to 31 nucleotides long and exist in most animals. They regulate the expression of transposons (jumping genes) by keeping the genes from being transcribed in the germ cells (sperm and eggs). Most piRNA are complementary to different transposons and can 19 | P r o f. D r. A s a l A z i z specifically target those transposons. Circular RNA (circRNA) is unique from other RNA types because its 5′ and 3′ ends are bonded together, creating a loop. The circRNAs are generated from many protein-encoding genes, and some can serve as templates for protein synthesis, similar to mRNA. They can also bind miRNA, acting as “sponges” that prevent miRNA molecules from binding to their targets. In addition, circRNAs play an important role in regulating the transcription and alternative splicing of the genes from which circRNAs were derived. Synthesis of RNA Synthesis of RNA is usually catalyzed by an enzyme—RNA polymerase—using DNA as a template, a process known as transcription. Initiation of transcription begins with the binding of the enzyme to a promoter sequence in the DNA (usually found "upstream" of a gene). The DNA double helix is unwound by the helicase activity of the enzyme. The enzyme then progresses along the template strand in the 3’ to 5’ direction, synthesizing a complementary RNA molecule with elongation occurring in the 5’ to 3’ direction. The DNA sequence also dictates where termination of RNA synthesis will occur. Primary transcript RNAs are often modified by enzymes after transcription. For example, a poly(A) tail and a 5' cap are added to eukaryotic pre-mRNA and introns are removed by the spliceosome. There are also a number of RNA- dependent RNA polymerases that use RNA as their template for synthesis of a new strand of RNA. For instance, a number of RNA viruses (such as poliovirus) use this type of enzyme to replicate their genetic material. 20 | P r o f. D r. A s a l A z i z RNA in disease Important connections have been discovered between RNA and human disease. For example, as described previously, some miRNAs are capable of regulating cancerassociated genes in ways that facilitate tumor development. In addition, the dysregulation of miRNA metabolism has been linked to various neurodegenerative diseases, including Alzheimer disease. In the case of other RNA types, tRNAs can bind to specialized proteins known as caspases, which are involved in apoptosis (programmed cell death). By binding to caspase proteins, tRNAs inhibit apoptosis; the ability of cells to escape programmed death signaling is a hallmark of cancer. Noncoding RNAs known as tRNA-derived fragments (tRFs) are also suspected to play a role in cancer. The emergence of techniques such as RNA sequencing has led to the identification of novel classes of tumor-specific RNA transcripts, such as MALAT1 (metastasis associated lung adenocarcinoma transcript 1), increased levels of which have been found in various cancerous tissues and are associated with the proliferation and metastasis (spread) of tumor cells. A class of RNAs containing repeat sequences is known to sequester RNA-binding proteins (RBPs), resulting in the formation of foci or aggregates in neural tissues. These aggregates play a role in the development of neurological diseases such as amyotrophic lateral sclerosis (ALS) and myotonic dystrophy. The loss of function, dysregulation, and mutation of various RBPs has been implicated in a host of human diseases. The discovery of additional links between RNA and disease is expected. Increased understanding of RNA and its functions, combined with the continued development of sequencing technologies and efforts to screen RNA and RBPs as therapeutic targets, are likely to facilitate such discoveries. 21 | P r o f. D r. A s a l A z i z  The Differences between DNA and RNA There are a number of differences that distinguish DNA from RNA: (a) RNA contains the sugar ribose, while DNA contains the slightly different sugar deoxyribose (a type of ribose that lacks one oxygen atom), (b) RNA has the nucleobase uracil while DNA contains thymine. (c) DNA is a double-stranded molecule that has a long chain of nucleotides. RNA is a single-stranded molecule which has a shorter chain of nucleotides. (d) DNA replicates on its own, it is self-replicating. RNA does not replicate on its own. (e) DNA and RNA molecules both contain four nitrogenous bases. Three of these (adenine, cytosine, and guanine) are found in both types of nucleic acid. (f) DNA molecules are self-replicating, whereas RNA molecules are synthesized by a process called transcription. (g) While DNA contains deoxyribose, RNA contains ribose, characterized by the presence of the 2′-hydroxyl group on the pentose ring (Figure 5). This hydroxyl group make RNA less stable than DNA because it is more susceptible to hydrolysis. (h) DNA and RNA molecules have different functions. DNA stores genetic information for the cell, whereas RNA codes for amino acids and acts as a messenger between DNA molecules and the ribosomes. 22 | P r o f. D r. A s a l A z i z Lecture No.4: Title of the lecture: DNA Organization in the cell DNA ORGANIZATION IN PROKARYOTIC CELLS A cell’s DNA, packaged as a double-stranded DNA molecule, is called its genome. In prokaryotes, the genome is composed of a single, double-stranded DNA molecule in the form of a loop or circle (Figure 4.1). The region in the cell containing this genetic material is called a nucleoid (remember that prokaryotes do not have a separate membrane-bound nucleus). Some prokaryotes also have smaller loops of DNA called plasmids that are not essential for normal growth. Bacteria can exchange these plasmids with other bacteria, sometimes receiving beneficial new genes that the recipient can add to their chromosomal DNA. Antibiotic resistance is one trait that often spreads through a bacterial colony through plasmid exchange. Figure (4.1): Bacterial DNA and plasmids are both circular. The size of the genome in one of the most well-studied prokaryotes, E. coli, is 4.6 million base pairs (which would be approximately 1.1 mm in length, if cut and stretched out). So how does this fit inside a small bacterial cell? The DNA is twisted by what is known as supercoiling. Supercoiled DNA is coiled more tightly than would be typically be found in a cell (more than 10 nucleotides per twist of the helix). If you visualize twisting a rope until it twists back on itself, you have a pretty good visual of supercoiled DNA. This process allows the DNA to be compacted into the small space inside a bacterium. 23 | P r o f. D r. A s a l A z i z DNA ORGANIZATION IN EUKARYOTIC CELLS Eukaryotes have much more DNA than prokaryotes. For example, an E. coli bacterium contains roughly 3 million base pairs of DNA, while a human contains roughly 3 billion. In eukaryotes such as humans and other animals, the genome consists of several double-stranded linear DNA molecules (Figure 4.2), which are located inside a membrane-bound nucleus. Each species of eukaryotes has a characteristic number of chromosomes in the nuclei (plural of nucleus) of its cells. A normal human gamete (sperm or egg) contains 23 chromosomes. A normal human body cell, or somatic cell, contains 46 chromosomes (one set of 23 from the egg and one set of 23 from the sperm; Figure 2). The letter n is used to represent a single set of chromosomes; therefore, a gamete (sperm or egg) is designated 1n, and is called a haploid cell. Somatic cells (body cells) are designated 2n and are called diploid cells. Figure (4.2): There are 23 pairs of homologous chromosomes in a female human somatic cell. The condensed chromosomes are viewed within the nucleus (top), removed from a cell in mitosis and spread out on a slide (right), and artificially arranged according to length (left); an arrangement like this is called a karyotype. In this image, the chromosomes were exposed to fluorescent stains for differentiation of the different chromosomes. A method of staining called “chromosome painting” employs fluorescent dyes that highlight chromosomes in different colors. 24 | P r o f. D r. A s a l A z i z Matched pairs of chromosomes in a diploid organism are called homologous (“same knowledge”) chromosomes. Of a pair of homologous chromosomes, one came from the egg and the second came from the sperm. Homologous chromosomes are the same length and have specific nucleotide segments called genes in exactly the same location, or locus. Genes, the functional units of chromosomes, determine specific characteristics by coding for specific proteins. Traits are the variations of those characteristics. For example, hair color is a characteristic with traits that are blonde, brown, or black. Each copy of a homologous pair of chromosomes originates from a different parent; therefore, the sequence of DNA present in the two genes on a pair of homologous chromosomes is not necessarily identical, despite the same genes being present in the same locations on the chromosome. These different versions of a gene that contain different sequences of DNA are called alleles. If you look at Figure (4.3), you can see a pair of homologous chromosomes. The chromosome shown in the figure is chromosome 15. The HERC2 gene is located on this chromosome. This gene is one of at least three genes that helps determine eye color. Each person inherits two copies of the HERC2 gene: one from the egg and one from the sperm. However, the alleles of the HERC2 gene that they inherit can be different. In the figure, the cell containing this homologous pair of chromosomes contains one blue allele and one brown allele. Figure (4.3): Homologous pair of chromosomes 15s, showing the location of HERC2 gene. Two different alleles of this gene are shown in either blue or brown. The variation of individuals within a species is due to the specific combination of the genes inherited from both parents. Even a slightly altered sequence of nucleotides within a gene can result in an alternative trait. 25 | P r o f. D r. A s a l A z i z For example, there are three possible gene sequences (alleles) on the human chromosome that code for blood type: sequence A, sequence B, and sequence O. Because all diploid human cells have two copies of the chromosome that determines blood type, the blood type (the trait) is determined by which two versions of the marker gene are inherited. It is possible to have two copies of the same allele on both homologous chromosomes, with one on each (for example, AA, BB, or OO), or two different alleles, such as AB. Minor variations of traits, such as blood type, eye color, and handedness, contribute to the natural variation found within a species. However, if the entire DNA sequence from any pair of human homologous chromosomes is compared, the difference is less than one percent. The sex chromosomes, X and Y, are the single exception to the rule of homologous chromosome uniformity: Other than a small amount of homology that is necessary to accurately produce gametes, the genes found on the X and Y chromosomes are different. EUKARYOTIC CHROMOSOMAL STRUCTURE AND COMPACTION If the DNA from all 46 chromosomes in a human cell nucleus was laid out end to end, it would measure approximately two meters; however, its diameter would be only 2 nm. Considering that the size of a typical human cell is about 10 µm (100,000 cells lined up to equal one meter), DNA must be tightly packaged to fit in the cell’s nucleus. At the same time, it must also be readily accessible for the genes to be expressed. During some stages of the cell cycle, the long strands of DNA are condensed into compact chromosomes. Eukaryotes, whose chromosomes each consist of a linear DNA molecule, employ a complex type of packing strategy to fit their DNA inside the nucleus (Figure 4). At the most basic level, DNA is wrapped around proteins known as histones to form structures called nucleosomes. The histones are evolutionarily conserved proteins that form an octamer of eight histone proteins attached together. DNA, which is negatively charged because of the phosphate groups, is wrapped tightly around the histone core, which has an overall positive charge. This nucleosome is linked to the next one with the help of a linker DNA. This is also known as the “beads on a string” structure. This is further compacted into a 30 nm fiber, which is the diameter of the structure. At the metaphase stage, the chromosomes are at their most compact, are approximately 700 nm in width, and are found in association with scaffold proteins. 26 | P r o f. D r. A s a l A z i z Figure (4.4): Double-stranded DNA wraps around histone proteins to form nucleosomes that have the appearance of “beads on a string.” The nucleosomes are coiled into a 30- nm chromatin fiber. When a cell undergoes mitosis, the duplicated chromosomes condense even further. DNA replicates in the S phase of interphase. After replication, the chromosomes are composed of two linked sister chromatids (Figure 4.5). This means that the only time chromosomes look like an “X” is after DNA replication has taken place and the chromosomes have condensed. During the majority of the cell’s life, chromosomes are composed of only one copy and they are not tightly compacted into chromosomes. When fully compact, the pairs of identically packed chromosomes are bound to each other by cohesion proteins. The connection between the sister chromatids is closest in a region called the centromere. The conjoined sister chromatids, with a diameter of about 1 µm, are visible under a light microscope. The centromeric region is highly condensed and thus will appear as a constricted area. In Figure 4, it is shown as an oval because it is easier to draw that way. Figure (4.5): Result of DNA replication in eukaryotes. In body cells, there are two copies of each chromosome (one from each parent). After replication, two identical sister chromatids remain connected at the centromere (shown as an oval). 27 | P r o f. D r. A s a l A z i z  Variation in DNA between Organisms Genetic variation is the presence of differences in sequences of genes between individual organisms of a species. It enables natural selection, one of the primary forces driving the evolution of life. Although each organism's DNA is unique, all DNA is composed of the same nitrogen-based molecules. So how does DNA differ from organism to organism? It is simply the order in which these smaller molecules are arranged that differs among individuals. Besides, the total percentage of GC varies over the range of 22-73%; such a variation places a difference in DNA sequences between individuals within a population. Variation occurs in germ cells i.e. sperm and egg, and also in somatic (all other) cells. 28 | P r o f. D r. A s a l A z i z However, DNA size is expressed in more than a way: 1- Number of base pairs 2- Molecular weight (1 base pair = 660 mw) 3- The length of 33.2 Å per helical turn of 10.4 base pairs 4- The DNA size is measured either using electron microscopes or gel electrophoresis Does the quantity of DNA vary in different organisms? The size, number of chromosomes, and nature of genomic DNA varies between different organisms (see table Sizes and molecular weights of various genomic DNAs). Viral DNA genomes are relatively small and can be single- or double-stranded, linear, or circular. 29 | P r o f. D r. A s a l A z i z Lecture No.5: The title of the Lecture: DNA Replication DNA replication The replication of the genome is essential for the continuity of life. The molecular mechanism is very similar in all groups of organisms. Although the basics of replication are already well understood, researchers are still focusing on questions relating to DNA replication. These questions not only deal with the understanding of a basic biological process, but also with related medical aspects. One attribute of living things is their ability to reproduce. The information required to pass on traits to the next generation is mainly stored in cellular DNA. Daughter and parent cells need to be equipped with an identical copy of DNA during cell division. “The molecular basis of this process is the replication of DNA”. However, in 1958, Matthew Meselson and Franklin Stahl showed that newly replicated bacterial DNA consists of a new and an old DNA strand. In the 1970s and 1980s, further evidence was found relating to the mechanism of ‘semiconservative’ replication. Replication consists of three phases: initiation, elongation and termination. The basis of replication is the pairing of the four bases found in DNA: adenine pairs with thymine, cytosine with guanine. The process, which results in two DNA helices instead of one, is mediated by several dozen proteins. However, the process is not the same at the end of the chromosomes, where a replication fork can no longer progress due to the lack of bases. At the site of the lagging strand where the DNA primase places the last RNA primer, the DNA polymerase is no longer able to continue replication. The terminal DNA segment cannot be replicated. The DNA strand gets shorter and shorter as the number of cell division rounds increases. So, to protect themselves against the rapid shortening of the DNA, eukaryotic chromosomes possess sequence repeats (telomeres) at their extremities, which do not code for proteins. Since the 30 | P r o f. D r. A s a l A z i z telomeres do not contain any important information, the key parts of the DNA are protected. The telomeres get shorter each time a cell divide. The length of these so-called telomere caps defines the number of possible divisions and hence the lifespan of a cell. Some cell types (for example maturing sex cells or certain tumor cells) contain the enzyme telomerase, which prevents telomere shortening and thus protects the cell from cell ageing and programmed cell death. Order in the cells A cell must at all costs prevent errors from occurring during the replication of DNA. The strict order of the copying process is therefore essential. In eukaryotic cells, the DNA is kept as clearly arranged as possible: genome regions that are not undergoing replication are densely packed in chromosomes. Scientists also assume that the chromosomes in eukaryotic cells are spanned over a cytoskeleton consisting of protein tubes and wires. The replication enzymes are bound to this so-called nuclear matrix and motor proteins pull the genome past them. It appears that bacterial cells have similar mechanisms. All in a good time Many bacteria divide once every thirty minutes, others replicate even faster. Eukaryotic cells only replicate their genome when new cells have to be created. This happens as a result of external signals, for example tissue loss or inflammation. The life cycle of eukaryotic cells underlies an accurately defined sequence of activities (cell cycle). However, any errors occur during the control of the cell cycle will coast cells to divide more quickly and more frequently as is the case with many cancer cells 31 | P r o f. D r. A s a l A z i z The process of DNA replication Replication is a complex process in which dozens of proteins, enzymes, and DNA structures take part; a single defective component can disrupt the whole process. The solution to this problem is central to replication. A huge amount of genetic information and an enormous number of cell divisions are required to produce a multicellular adult organism; even a low rate of error during copying would be fatal. How is this extraordinarily accurate and rapid process accomplished? 1- The complementary nature of the two nucleotide strands in a DNA molecule suggested that, during replication, each strand can serve as a template for the synthesis of a new strand. 2- 2- The specificity of base pairing (adenine with thymine; guanine with cytosine) implied that only one sequence of bases can be specified by each template, and so two DNA molecules built on the pair of templates will be identical with the original. 3- Eukaryotic DNA is replicated in more than replication foci. (a) DNA Synthesis Begins at Replication Origins The process of DNA replication begun by initiator proteins that bind to the DNA and separate the two strands apart, breaking the hydrogen bonds between the bases. The positions at which the DNA is first opened are called replication origin. They are usually marked by a particular sequence of nucleotides figure (4.1). Figure (4.1): Schematic diagram of origin of replication in human. 32 | P r o f. D r. A s a l A z i z  (b) DNA Synthesis Occurs at Replication Forks During the DNA replication it is possible to see Y-shaped junctions in the DNA, called “the replication forks”. See figure (4.2) At these forks, the replication machine is moving along the DNA, opening up the two strands of double helix and using each strand as a template to make new daughter strand. Two replication forks are formed starting from each replication origin, and they move away from the origin in both directions, unzipping DNA as they go. Figure (4.2): Simple replication fork (c) Elongation of a new strand Elongation of new DNA at replication fork is catalyzed by enzymes called DNA polymerase. As nucleotides align with complementary bases along “old” template strand of DNA, they are added by polymerase, one by one, to the growing end of the new DNA strand. The rate of elongation is about 50 nucleotides per second in human cells. As each monomer joins the growing end of DNA strand, it losses two phosphate groups. Hydrolysis of phosphate is the exergonic reaction that drives polymerization of nucleotides to from DNA. Replication Requirements Although the process of replication includes many components, there are four components very essential for the process, they include: - 1. A template consisting of single-stranded DNA;( in eukaryotes, the replication fork forms at the replication initiation point (RIP) where the helicase enzymes unwinds the DNA double helix. The enzyme DNA polymerase, situated at each replication fork, builds a new DNA strand (template) by adding nucleotides in the 5' to 3' 33 | P r o f. D r. A s a l A z i z direction. Also performs proof-reading and error correction. DNA polymerase replicates a DNA template with remarkable fidelity. 2. Substrates to be assembled into a new nucleotide strand; the four types of the deoxyribonucleotide – triphosphate (dCTP, dTTP, dGTP, dATP). These substrates are provided from two pathways for nucleotide biosynthesis (the de novo pathway: nucleotides are constructed from simple precursors and the salvage pathways: recovery and recycling of nucleotides obtained in the diet). *In humans, dietary nucleotide bases are rarely incorporated into nucleotides. As a result, humans must synthesize their own nucleotide bases. (With the exception of a few parasitic prokaryotes, all organisms can synthesize nucleotides.) Although all nucleated eukaryotic cells can synthesize nucleotides, most human synthesis occurs in the liver. Nucleotide synthesis is tightly regulated. Nucleotide synthesis is somewhat expensive in that the pathways use several molecules with other uses. In addition, although pyrimidines can be degraded into standard metabolic intermediates, purine catabolism does not alter the basic purine structure, and excessive levels of purines can be toxic. 3. Enzymes including (DNA helicases- unwinds the double helix, DNA polymerase- build a new DNA and performs proof- reading error correction, primase- provide a primer and DNA ligase that joins the Okazaki fragments together) and other proteins that “read” the template and assemble the substrates into a DNA molecule. 4. Primer (short sequence of DNA or RNA) in the template with a free –OH end. This short sequence is provided by the primase enzyme to provide a starting point for the DNA polymerase to begin synthesis of the new DNA strand. The Mechanism of Replication DNA replication takes place in four stages: initiation, unwinding, elongation, and termination, illustrated in (Figure 4.3). 1- Initiation of replication Eukaryotic cells utilize thousands of replication origins due to the large size of their genome, and so the entire genome can be replicated in a timely manner. The use 34 | P r o f. D r. A s a l A z i z of multiple origins, however, creates a special problem in the timing of replication: the entire genome must be precisely replicated once and only once in each cell cycle so that no genes are left unreplicated and no genes are replicated more than once. How does a cell ensure that replication is initiated at thousands of origins only once per cell cycle? The precise replication of DNA is accomplished by the separation of the initiation of replication into two distinct steps: - ◄ -In the first step, the origins are licensed, meaning that they are approved for replication. This step is early in the cell cycle when a replication licensing factor attaches to an origin. ◄In the second step, initiator proteins cause the separation of DNA strands and the initiation of replication at each licensed origin. The key is that initiator proteins function only at licensed origins. As the replication forks move away from the origin, the licensing factor is removed, leaving the origin in an unlicensed state, where replication cannot be initiated again until the license is renewed. To ensure that replication takes place only once each cell cycle, the licensing factor is active only after the cell has completed mitosis and before the initiator proteins become active. 2. Unwinding Several helicases that separate double-stranded DNA have been isolated from eukaryotic cells, as have single strand- binding proteins and topoisomerases. These enzymes and proteins are assumed to function in unwinding eukaryotic DNA. 3. Elongation Eukaryotic cells contain a number of different DNA polymerases that function in replication, recombination, and DNA repair. However, through the elongation process, the DNA polymerase α _, which contains primase activity, initiates nuclear DNA synthesis by synthesizing RNA primer, followed by a short string of DNA nucleotides. After DNA polymerase _ has laid down from 30 to 40 nucleotides, DNA polymerase δ _ completes replication on the leading and lagging strands. DNA polymerase β _ does not participate in replication but is associated with the repair and recombination of nuclear DNA. 35 | P r o f. D r. A s a l A z i z  However, the following concepts review the mechanism of eukaryotic replication: 1. Replication is always semiconservative. 2. Replication begins at sequences called origins. 3. DNA synthesis is initiated by short segments of RNA called primers synthesized by primase enzyme to make a template with free 3- OH end to be used by the DNA polymerase that add nucleotides in the new strands. 4. The elongation of DNA strands is always in the (5´→3´) direction. 5. New DNA is synthesized from dNTPs; in the polymerization of DNA, two phosphates are cleaved from a dNTP and the resulting nucleotide is added to the 3-OH group of the growing nucleotide strand. 6. Replication is continuous on the leading strand and discontinuous on the Lagging strand to create Okazaki fragments, these fragments are made of 1000 nucleotides, after replication stops, these fragments undergo excision process where DNA polymerase removes the primers from these fragments and are ligated by DNA ligase together to form an elongated strand complementary to their template. 7. New nucleotide strands are made complementary and antiparallel to their template strands. 8. Replication takes place at very high rates and is accurate, due to Precise nucleotide selection, proofreading, and repair mechanisms. 4. Termination In some DNA molecules, replication is terminated whenever two replication forks meet. In others, specific termination sequences block further replication. 36 | P r o f. D r. A s a l A z i z  The replication of cancer cells – A target for therapy Replication is also of great interest in the field of medicine, in particular in fighting against cancer. Cancer cells are body cells that no longer behave normally - they replicate their genome and proliferate far more often than healthy cells. Researchers and physicians exploit this behavior in their work on substances designed to interfere with cancer growth. Some substances inhibit replication, which prevents tumor growth. Modern chemotherapy uses alkylating agents (e.g., busulfan, Ifex). Busulfan is used to treat chronic myelogenous leukemia (CML). It does not cure the disease but helps to control it so that your quality of life is improved. Ifex is used to treat various cancers (such as testicular cancer). It works by slowing or stopping the growth of cancer cells. These substances bind to DNA via alkyl groups. Since these groups have two binding sites, the genome is joined together, thereby preventing it from replicating. Another example is platinum analogues that are among the most effective chemotherapeutic drugs. These substances have a platinum atom that binds to the DNA and joins it (e.g., cisplatin, carboplatin). They make the DNA un accessible to DNA polymerase, thereby preventing the enzyme from synthesizing the DNA of the cancer cell. 37 | P r o f. D r. A s a l A z i z Lecture No.6: Title of the lecture: DNA Transcription & Post transcriptional Modification DNA Transcription Since the food that enters our bodies cannot be used as it is. Thus, it should go through a number of chemical processes to be digested. Thereby, the chemical processes in our stomach uses different proteins and enzymes to break down the food particles into usable nutrients our cells can absorb. All of the instructions needed for these proteins to be manufactured are stored in our DNA. The DNA contain genes which are a string of nucleotides that encode for the information required for protein in a process called (Gene expression). The process of gene expression is divided into two processes (transcription and translation). In eukaryotic cell the process is carried out in the nucleus. DNA transcription requires that sequences on DNA are accessible to RNA polymerase and other proteins. However, to achieve this; the chromatin structure is modified before transcription so that; the DNA is in a more open configuration and is more accessible to the transcription machinery. The transcription process occurs in three steps: 1- Initiation 2- Elongation 3- Termination 38 | P r o f. D r. A s a l A z i z During initiation, the promoter region of the gene acts as a recognition site for RNA polymerase to bind. Figure 5.1 ((this is where the majority of gene expression is controlled by either permitting or blocking the access of RNA polymerase to this site. Moreover, another type of controlling elements also plays an important role in the transcription initiation; they are called ((the enhancers)). Figure (5.1): Initiation in DNA Transcription An enhancer, it can affect the transcription of genes that are thousands of nucleotides away, and their positions relative to start sites can vary. See figure 5.2. Figure (5.2): Enhancer driven initiation of Transcription. Binding of RNA polymerase to this site cause the DNA double helix to unwind and open. Thereby, during elongation step. 39 | P r o f. D r. A s a l A z i z In the elongation step, the RNA polymerase slides along the template DNA strand. As the complementary bases pair up in the 5 to 3 direction, the DNA transcription elongation is in the direction of 5´ → 3 ´ and Transcription begins at the start site, which is determined, the consensus sequences. A short stretch of DNA is unwound near the start site and used as a template this is called “the sense strand”, it determines the amino acid sequence in the produced protein. On the other hand, the other strand is called “the antisense strand” that is not transcribed into mRNA but it is used as a template in the DNA replication for the synthesis of the sense strand. figure 5.3. Figure (5.3): Elongation in DNA Transcription Once RNA polymerase reaches to the termination site of the gene. Then, RNA polymerase, the DNA molecule and the messenger RNA dissociate from each other pronouncing the termination of the transcription see figure 5.4. Figure (5.4): Elongation in DNA Transcription 40 | P r o f. D r. A s a l A z i z However, three types of RNA polymerase transcribe different RNA molecules. 1- RNA polymerase I = rRNA 2- RNA polymerase II = mRNA 3- RNA polymerase III = tRNA + rRNA consequently, RNA molecule will result from the transcription process that differs between prokaryotes, figure 5.6 and eukaryotes as figure 5.7 showed. Figure (5.): DNA Transcription in prokaryotes The resulting mRNA in eukaryotes is called ((pre-mRNA)) because it contains ((Exons – the protein coding regions)) and ((Introns- the non-coding regions)). This primary transcript needs some processing to become mature mRNA. See figure 5.7 Figure (5.7): DNA Transcription in eukaryotes 41 | P r o f. D r. A s a l A z i z  Post transcriptional modification Moreover, the produced primary transcript (pre-mRNA) is not ready to be transformed to the ribosomes for translation. Yet, it should be subjected to the “Post – transcriptional modifications”. ((Post Transcriptional Modification)) Which comprises: - 1. The addition of “Cap” This process, known as mRNA capping, is highly regulated and vital in the creation of stable and mature messenger RNA able to undergo translation during protein synthesis. Capping is accomplished through the unusual 5′ to 5′ triphosphate linkage of a guanine nucleotide to mRNA. This guanine is methylated on the 7´ position directly after capping by a methyl transferase. It is referred to as a 7-methylguanylate cap. The 5′ cap has four main functions: a. Regulation of nuclear export b. Prevention of degradation by exonucleases c. Promotion of translation d. Promotion of intron excision 42 | P r o f. D r. A s a l A z i z 2. Polyadenylation is the addition of a poly (A) tail to a messenger RNA. It consists of multiple adenosine monophosphates. In eukaryotes, this process protects the mRNA molecule from enzymatic degradation in the cytoplasm and aids in transcription termination, export of the mRNA from the nucleus, and translation. See (figure 5.8). 3. The excision of Introns This process is called (Intron splicing) and is performed by a complex made of proteins and RNA called (a spliceosome). This complex removes the intron segments and join the adjacent exons to produce mature mRNA strand that can leave the nucleus through a nuclear pore and enter the cytoplasm to begin translation. Figure (5.8): Summary of Eukaryotic DNA Transcriptional modification and mRNA processing. 43 | P r o f. D r. A s a l A z i z Lecture No7: Title of the lecture: DNA Translation How does the cell convert DNA into working proteins? As you Knew before, the genes in our DNA encode for protein molecules, which are the "work horses" of the cell, carrying out all the functions necessary for life. For example, enzymes, including those that metabolize nutrients and synthesize new cellular constituents, as well as DNA polymerases and other enzymes that make copies of DNA during cell division, are all proteins. During translation, which is the second major step in gene expression, the mRNA is "read" according to the genetic code which relates the DNA sequence to the amino acid sequence in proteins. Each group of three bases in mRNA constitutes a codon (every three bases specify a codon) and each codon specifies a particular amino acid where the genetic code includes ((64 codons)). The mRNA sequence is thereby used as a template to assemble—in order—the chain of amino acids that form a protein where there are four special codons in the genetic code; one codes for Start (AUG) and three code for Stop (UGA, UAG, UAA). Where Translation Occurs? Within all cells, the translation machinery resides within a specialized organelle called the ribosome. In eukaryotes, mature mRNA molecules must leave the nucleus and travel to the cytoplasm, where the ribosomes are located. Eukaryotic ribosomes have two unequal subunits, designated small subunit (40S) and large subunit (60S) according to their sedimentation coefficients. Both subunits contain dozens of ribosomal proteins arranged on a scaffold composed of ribosomal RNA (rRNA). See (figure 6.1). 44 | P r o f. D r. A s a l A z i z  Figure (6.1): Eukaryotic 80S ribosome How many ribosomes are in eukaryotes? A single actively replicating eukaryotic cell, for example, may contain as many as 10 million ribosomes. In the bacterium Escherichia coli (a prokaryote), ribosomes may number as many as 15,000, constituting as much as one-quarter of the cell's total mass Why does 60S and 40S make 80S? The 80S ribosome is identified according to its sedimentation coefficients in Svedberg units. Since it is a measure of sedimentation time, it is not simply a sum of subunits. Svedberg units are a nonlinear function, and 60S and 40S are two separate subunits that together sediment at 80S On the other hand, Bacteria and archaebacteria have smaller ribosomes, termed 70S ribosomes in their cytoplasm, which are composed of a small 30S subunit and large 50S subunit. The "S" stands for Svedberg’s, a unit used to measure how fast molecules move in a centrifuge. What is difference between 70S and 80S? The 70S ribosomes are smaller and have a simpler structure than the 80S ribosomes. The 70S ribosomes are composed of a small number of proteins and RNA molecules, while the 80S ribosomes are composed of a larger number of proteins and RNA molecules. The 80s ribosomes are also more stable than the 70S ribosomes. Figure (6.2). Figure (6.2): Eukaryotic 80S ribosome VS Prokaryotic 70S ribosome 45 | P r o f. D r. A s a l A z i z Still, each subunit of the ribosomes exist separately in the cytoplasm, but the two join together on the newly transcribed mRNA molecule in order to start translation. Translation consists of three steps: 1- Initiation 2- Elongation 3- Termination 1- Initiation Translation begins when the mRNA strand binds to the small ribosomal subunit upstream the start codon, “AUG” start codon. See figure below. Figure (6.3): Initiation of translation Interestingly, the “start codon- AUG” alone is not enough to initiate translation, it needs other factors to initiate the translation chain whether in eukaryotes or in prokaryotic cells. In eukaryotes, there is an area near the 5' end of the molecule that is known as the untranslated region (UTR) or leader sequence or more specifically called “Kozak box”. Figure (6.4): Kozak sequence 46 | P r o f. D r. A s a l A z i z The Kozak sequence is named after the scientist Marilyn Kozak who discovered it. It is a nucleic acid motif that initiates protein translation in eukaryotic mRNA. A start codon is located within the Kozak sequences where the assembly of ribosomes begins. What is the Kozak sequence in human cells? The Kozak sequence directs the pre-initiation complex (PIC) and ribosome to the translation initiation site (start codon) and mediates ribosome assembly ensuring the correct protein sequence is translated. The consensus Kozak sequence is generally considered as GCCGCCACCATGG, where ATG is the start codon. Is Kozak sequence only in eukaryotes? The scanning mechanism of initiation, which utilizes the Kozak sequence, is found only in eukaryotes and has significant differences from the way bacteria initiate translation. This portion of mRNA is located between the start codon (AUG) and the first nucleotide that is transcribed in the coding region, and it does not affect the sequence of amino acids in a protein. So, what is the purpose of the UTR? It turns out that, the leader sequence is important because it is a protein translation initiation site found on the mRNA of eukaryotes. On the other hand, in bacteria, this site is known as the Shine-Dalgarno box (AGGAGG), after scientists John Shine and Lynn Dalgarno who first characterized it. Figure (6.5): Shine-Dalgarno box The Shine–Dalgarno (SD) sequence motif facilitates translation initiation and is frequently found upstream of bacterial start codons. 47 | P r o f. D r. A s a l A z i z The translation of mRNA begins with the formation of a complex on the mRNA where, three initiation factor proteins (known as IF1, IF2, and IF3) bind to the small subunit of the ribosome (40S) Then, a methionine-carrying tRNA will bind to the mRNA, near the AUG start codon, forming the initiation complex. ► Although methionine (Met) is the first amino acid incorporated into any new protein; it is not always the first amino acid in mature proteins. In many proteins, methionine is removed after translation. In fact, if a large number of proteins are sequenced and compared with their known gene sequences. However, each amino acid is brought to the ribosome by a specific tRNA molecule and the type of amino acid depends on the anticodon sequence of the tRNA molecule where a complementary occurs between the codon of the mRNA and the anticodon of the tRNA. After the initiation tRNA binds to the mRNA strand, the large rRNA subunit (60S) binds to the mRNA to form the translation complex and the ignition then is completed. 2- Elongation In the large ribosomal subunit, there are three distinct regions; called (E, P, A) sites. See figure (6.6). Figure (6.6): Ribosome structure Where, E= Exit site P= Peptide bond- tRNA binding site A= Amino acid tRNA binding site During elongation, individual amino acids are brought to the mRNA strand by tRNA molecule through complementary base pairing between the codons (in mRNA) and the anticodons (in tRNA) where it corresponds to a particular amino acid. Elongation occurs in these steps: Figure (6.7) 1- A charged tRNA molecule binds to the A site and a peptide bond forms between its amino acid and the one of the initiators tRNA at the P site. 2- The complex slides one codon to the right where the uncharged tRNA exits from the E site. 4- The A site is open now to accept a new charged tRNA molecule according to the codon of the mRNA and Elongation continues until a stop codon is reached. 48 | P r o f. D r. A s a l A z i z Figure (6.7): Elongation in translation 3- Termination A release factor binds to the A site at a stop codon and the polypeptide is released from the tRNA in the P site and the entire complex dissociates and can reassemble to begin the process again where the purpose of translation is to produce polypeptides quickly and accurately. Figure (6.8): Termination of translation 49 | P r o f. D r. A s a l A z i z  The difference between prokaryotes and eukaryotes in translation Diff. Prokaryotes Eukaryotes 1 It is a continuous process as both It is a discontinuous process as transcription and translation transcription occurs in the nucleus and processes occur in the cytoplasm translation occurs in the cytoplasm 2 Little mRNA processing Extensive mRNA processing of three steps 3 Polycistronic mRNA Monocistronic mRNA (synthesis information of many (synthesis information of only one proteins under a single control) protein under a single control) 4 It occurs on 70S ribosomes It occurs on 80S ribosomes 5 Ribosome small subunit binds to Translation initiation occur at Kozak box Shine Dalgarno sequence 6 Initiator tRNA is (fMet/tRNA) Initiator tRNA is (Met/tRNA) which which codes for formyl methionine codes for methionine 7 Simple initiation of translation Complex initiation process involves many factors 8 Termination involves the release of Only one release factor recognizes all many factors three stop codons 9 Low number of post transcriptional Extensive post translational modifications occur in the modifications occur in the Endoplasmic cytoplasm reticulum and Golgi apparatus before becoming a fully functional protein 10 mRNA mRNA half life is short (few seconds) half life is longer (few hours to few days) unstable quite stable 11 It is a faster process Comparatively slower Add 17-21 amino acids /sec. Add 6-9 amino acids/sec. Protein Synthesis Protein synthesis is the process whereby biological cells generate new proteins; and the process is balanced by the loss of cellular proteins via degradation or export and the need to a new protein. This process is accomplished through two processes (DNA transcription and Translation); where the translation process plays an essential part of the biosynthetic pathway. 50 | P r o f. D r. A s a l A z i z Post translation modifications After dissociation, the polypeptide might need to be modified before it is ready to function. Modifications takes place in different organelles for different proteins. For example: in order for a digestive enzyme to be secreted into the stomach, the polypeptide is translated into the endoplasmic reticulum, modified as it passes through ((Golgi apparatus)), then secreted using a vesicle through the plasma membrane of the cell into the lumen of the digestive tract. Thus, the ((Post-translational modification (PTM)) refers to the covalent and generally enzymatic modification of proteins following protein biosynthesis. PTMs are important components in cell signaling, as for example when prohormones are converted to hormones. Post-translational modifications can occur on the amino acid side chains or at the protein's C- or N- termini. They can extend the chemical repertoire of the 20 standard amino acids by modifying an existing functional group or introducing a new one such as phosphate. Significance of studying PTMs: Addiction A major feature of addiction is its persistence. The addictive phenotype can be lifelong, with drug craving and relapse occurring even after decades of abstinence. Yet, post-translational modifications in addiction involve epigenetic alterations of histone protein tails in specific regions of the brain of the addictive person. Once particular post-translational epigenetic modifications occur, they appear to be long lasting "molecular scars" that may account for the persistence of addictions. 51 | P r o f. D r. A s a l A z i z Inhibitors of translation A translation or a protein synthesis inhibitor is a substance that stops or slows the growth or proliferation of cells by disrupting the processes that lead directly to the generation of new proteins. However, there are numbers of antibiotics act by inhibiting translation. These include clindamycin, anisomycin, cycloheximide, chloramphenicol, tetracycline, streptomycin, erythromycin, and puromycin. Prokaryotic ribosomes have a different structure from that of eukaryotic ribosomes, and thus some of these antibiotics can specifically target bacterial infections without any harm to a eukaryotic host's cells. On the other hand, a natural product, namely (nagilactone C) produced from Podocarpus trees, were identified and characterized as novel protein synthesis inhibitors in humans and animals. This compound is specific for the eukaryotic translation apparatus inhibition and interfere with translation elongation. Still, since protein synthesis plays an essential role in the cell proliferation, differentiation, and survival. Inhibitors of eukaryotic translation have entered the clinic, establishing the translation machinery as a promising target for chemotherapy. A recently discovered, structurally unique marine sponge-derived brominated alkaloid, agelastatin A (AglA), possesses potent antitumor activity. 52 | P r o f. D r. A s a l A z i z Lecture No.8: Title of the lecture: Gene Expression and Regulation Expression Gene expression term refers to the process of converting the genetic information stored in the gene into a functional protein. This process is accomplished throughout two basic stages in both eukaryotes and prokaryotes; named (transcription and translation) where the genetic information present in the DNA are transcribed into mRNA molecule and then, translated into proteins by the ribosomes. At the simplest level, a gene could be defined as a unit of information that encodes a genetic characteristic. It is about a specific nucleotide sequences present in the DNA molecule representing the genetic unit in living organisms. However, there are differences in gene expression between prokaryotes and eukaryotes where prokaryotic gene expression (both transcription and translation) occurs within the cytoplasm of a cell due to the lack of a defined nucleus; thus, the DNA is freely located within the cytoplasm. While, Eukaryotic gene expression occurs in two sites; the nucleus (transcription) and cytoplasm (translation). Moreover, since the genetic information is encoded in the molecular structure of the DNA (genes). The difference in gene arrangement between prokaryotes and eukaryotes was also situated considering gene expression: In prokaryotes, the genome is composed of a single, double-stranded DNA molecule in the form of a loop or circle. The region in the cell containing this genetic material is called a nucleoid. Some prokaryotes also have smaller loops of DNA called plasmids that are not essential for normal growth. In addition, the prokaryotic gene structure consists of operons and clusters of several functionally-related genes, whereas the eukaryotic gene structure does not contain operons. Figure (7.1) 53 | P r o f. D r. A s a l A z i z  Figure (7.1): Gene structure in prokaryotes While, in eukaryotes, the store house of genetic information within the cell are chromosomes, which consist of DNA and associated proteins, figure (7.2). The cells of each species have a characteristic number of chromosomes each carries a large number of genes. Many genes encode traits by specifying the structure of proteins. Genetic information is first transcribed from DNA into RNA, and then RNA is translated into the amino acid sequence of a protein. In a process called “the Central Dogma of Life”. Figure (7.2): Gene assembly in eukaryotes Characteristics of Eukaryotic gene structure: All genes contain a coding region, which is split on the upstream of what is conventionally called promoter which is (by definition, the site for the assembly of transcriptional apparatus and its accessory factors, where they bind to it and open the DNA to initiate transcription) see figure (7.3). Figure (7.3): Organization of eukaryotic gene region 54 | P r o f. D r. A s a l A z i z The genomes of most eukaryotes are larger and more complex than those of prokaryotes (Figure 4.1). This larger size of eukaryotic genomes is not inherently surprising, since one would expect to find more genes in organisms that are more complex. However, the genome size of many eukaryotes does not appear to be related to genetic complexity. For example, the genomes of salamanders and lilies contain more than ten times the amount of DNA that is in the human genome. There are however, certain regions in the DNA which contribute to the large size of DNA: 1- large amounts of noncoding DNA are also found within most eukaryotic genes. Such genes have a split structure in which segments of coding sequence (called exons) are separated by noncoding sequences (intervening sequences, or introns). 2- Another factor contributing to the large size of eukaryotic genomes is that some genes are repeated many times. Whereas most prokaryotic genes are represented only once in the genome, many eukaryotic genes are present in multiple copies, called gene families. 3- A substantial portion of eukaryotic genomes consists of highly repeated noncoding DNA sequences. These sequences, sometimes present in hundreds of thousands of copies per genome 4- Other repetitive DNA sequences are scattered throughout the genome rather than being clustered as tandem repeats. These sequences are classified as SINEs (short interspersed elements) or LINEs (long interspersed elements). In the same context, in eukaryotes, structural genes are not sequentially placed. Each gene is instead composed of coding exons and interspersed non-coding introns. Regulatory sequences are typically found in non-coding regions upstream and downstream from the gene. As figure (7.3) showed, Outside the transcriptional area, the 5' flanking region ("upstream") gene region includes the 'CAT box' and 'TATA box' promoters required for RNA polymerase recognition prior to transcription. Enhancers that regulate occurrence, timing, and amount of transcription occur in both the upstream region and the 3' 55 | P r o f. D r. A s a l A z i z flanking region ("downstream") region; multiple enhancers may occur many hundreds of nucleotides upstream. Figure (7.4): Schematic representation of eukaryotic gene The components of the promoter, depending upon, which acts as first recognition sequence for the assembly of RNA-polymerase complex, without which enzymes won’t assemble. The start point itself is bracketed by a set of sequences called “the TATA box” which is a sequence within the promoter core. Besides, there are other factors binding to specific sequence boxes increase the efficiency of transcription depending on the type of RNA polymerase enzyme as Eukaryotes have three different classes of RNA polymerases, such as RNA polymerase I, RNA polymerase II and RNA polymerase III and each of them transcribe specific groups of RNAs, like rRNA genes and tRNA gene. The beginning and the end of each structural gene is linked to a specific sequence of nucleotides called “the control elements” that participate in the regulation of the transcription process through their reaction with the RNA polymerase and the other regulatory proteins. The other most important control element is called “the terminator” that is located downstream of the structural gene which signals the RNA polymerase to detach from the DNA template and terminate the transcription process. Though, structural genes in eukaryotes comprise the majority of the genes present in the chromosomes. However, eukaryotic cells also include other types of genes that their product is RNA molecules other than proteins; such as structural genes responsible for the assembly of” rRNA”, others are responsible for the creation of” tRNA” which both play an important role in the translation of the transcribed mRNA. Therefore, the components of the promoter in both prokaryotes and eukaryotes vary as figure (7.5) shows where many factors and components acts as first recognition sequence for the assembly of RNA-polymerase complex, without which this crucial enzyme won’t assemble and no gene expression products. 56 | P r o f. D r. A s a l A z i z  Figure (7.4): Schematic representation of eukaryotic gene The components of the promoter, depending upon, which acts as first recognition sequence for the assembly of RNA-polymerase complex, without which enzymes won’t assemble. The start point itself is bracketed by a set of sequences called “the TATA box” Besides the binding of RNA polymerase complex to TATA box, other factors binding to specific sequence boxes increase the efficiency of transcription. The promoter components vary depending upon the type of the RNA polymerase that is involved. Eukaryotes have three different classes of RNA polymerases, such as RNA polymerase I, RNA polymerase II and RNA polymerase III and each of them transcribe specific groups of RNAs, like rRNA genes and tRNA gene. Table 1: Differences in the Regulation of Gene Expression of Prokaryotic and Eukaryotic Organisms Prokaryotic organisms Eukaryotic organisms Lack nucleus Contain nucleus RNA transcription occurs prior to protein translation, and it takes place in RNA transcription and protein translation the nucleus. RNA translation to protein occurs in the cytoplasm. occur almost simultaneously RNA post-processing includes addition of a 5′ cap, poly-A tail, and excision of introns and splicing of exons. Gene expression is regulated primarily at Gene expression is regulated at many levels (epigenetic, transcriptional, the transcriptional level post-transcriptional, translational, and posttranslational) 57 | P r o f. D r. A s a l A z i z Gene Regulation in Eukaryotic cells Gene regulation in eukaryotic cells may occur before or during transcription or translation or even after protein synthesis. Regulation of gene expression involves many different mechanisms where in ((Multicellular organisms)) it is more complicated because of the large genome and the presence of a nucleus within the cytoplasm that provide a more compartmentalized structure. However, there are a number of different stages at which gene expression may be regulated in eukaryotes: 1. The Process of “chromatin” Remodeling In the nucleus, the process of chromatin remodeling regulates the availability of a gene for transcription where several gene expression mechanisms involve the remodeling of chromatin structure. When eukaryotic cells aren't dividing, chromosomes exist in an uncondensed state called chromatin; which consists of DNA wrapped around a histone protein core. The wrapped DNA isn't as available for transcription by RNA polymerase directly, but instead it binds via a set of proteins: (the transcription initiation complex). In accordance, there are two different types of chromatin can be seen during interphase: they are called euchromatin and heterochromatin. Figure (7.5): Schematic representation of Euchromatin VS Heterochromatin in eukaryotic gene As the figure above shows, Euchromatin, which is a lightly packed form, contains areas of DNA that are undergoing active gene transcription. Not all of the chromatin is undergoing gene transcription, however. Heterochromatin, in contrast, is mostly inactive DNA that is being actively inhibited or repressed in a region-specific manner. The chromatin state can change in response to cellular signals and gene activity. This is facilitated by enzymes that modify histones by adding methyl and acetyl groups to their N-terminal tails. 58 | P r o f. D r. A s a l A z i z 2. Transcription is an important stage for gene regulation. The RNA polymerase needs transcription factors to initiate transcription. These factors bind to proximal or distal control elements, which are specific DNA sequences that are usually four to eight base pairs long. The rate of gene expression may be greatly affected by binding of specific transcription factors to control elements. Proximal control elements are close to the promoter. Distal control elements may be grouped as enhancers, and may be thousands of nucleotides removed from the gene. How does the binding of transcription factors to control elements regulate transcription? There seem to be two structural components in transcription factors: a region that binds to DNA and an activation domain that attaches to other proteins or components of the transcription apparatus itself. There are only a few different kinds of binding regions in control elements: these are called DNA sequence motifs. The binding of transcription factors that function as activators to control elements in an enhancer may cause the DNA to bend. This bending brings the enhancer complex into contact with the protein complex at the proximal promoter, creating a large complex that promotes RNA polymerase 59 | P r o f. D r. A s a l A z i z binding. RNA polymerase II is then recruited and transcription can begin. Some transcription factors function as repressors that bind to control elements, effectively blocking the binding of activators. 3. Additional mechanisms of gene expression Occur after transcription One post-transcriptional control mechanism is alternative RNA splicing, which produces different mRNA molecules from the same primary transcript. The primary mRNA transcript is composed of both non-coding introns and protein- coding exons. Regulatory proteins remove the introns and control the exon choices by binding to regulatory sequences within the primary transcript. Alternative RNA splicing may be a major reason for the diversity of proteins in higher animals over those found in simpler organisms. For example, the number of genes in a human is remarkably similar to those in a nematode or sea urchin. However, there are many more genes with multiple exons in higher animals, so alternative RNA splicing may provide a way of making more types of proteins from the same amount of genomic DNA. Eukaryotic mRNA molecules are longer-lasting than prokaryotic mRNAs, and mRNA life span is a key parameter in protein synthesis in a cell. Rapid mRNA degradation is a useful feature of prokaryotes because they may need to change their protein synthesis rapidly in response to environmental changes. In eukaryotes, some mRNAs may exist for periods ranging from days to weeks, and they may be repeatedly translated, such as the mRNAs that produce hemoglobin molecules in red blood cells. mRNA stability seems to be associated with changes in the poly(A) tail length. If the tail is shortened, enzymes may be triggered that remove the 5′ cap of the RNA. Once the cap is removed, nuclease enzymes degrade the mRNA. 60 | P r o f. D r. A s a l A z i z 4. Regulation of gene expression during translation Before a polypeptide is produced, translation has to occur. Therefore, another stage where control of gene expression can occur is by blocking the initiation of translation. One place where this is often seen is in unfertilized eggs. Eggs have many mRNA molecules that are not translated until fertilization occurs. These mRNAs will produce many proteins that are important in development, but they are not needed while the egg waits to be fertilized. Translation of the mRNAs may be blocked by the binding of regulatory proteins to sequences in the leader region at the 5′ end of the mRNA (the Kozak box), preventing the attachment of ribosomes. Finally, regulation occur post- translationally. In eukaryotes, polypeptides must often be processed to create functional proteins. For example, proteins may need to be folded, modified by adding carbohydrate groups, or activated by phosphorylation. Regulation may occur by modifying any of these steps 61 | P r o f. D r. A s a l A z i z Lecture No.9: Title of the Lecture: The Genetic code and It’s applications The Genetic code The genetic code is the code our body uses to convert the instructions contained in our DNA the essential materials of life. It is typically discussed using the “codons” found in mRNA, as mRNA is the messenger that carries information from the DNA to the site of protein synthesis. What is the definition of the genetic code? Genetic code refers to the instructions contained in a gene that tell a cell how to make a specific protein. What is genetic code and its types? The genetic code is of two types. The genetic code can be expressed as either RNA codons or DNA codons. RNA codons occur in messenger RNA (mRNA) and are the codons that are actually “read” during the synthesis of polypeptides (the process called translation). 62 | P r o f. D r. A s a l A z i z How is genetic code created? The genetic code is made up of codons, which are three-letter chains of nucleotides. Each codon codes for one specific amino acid. The code determines the order in which amino acids are added to a polypeptide chain during protein synthesis. Therefore, the genetic code dictates the sequence of amino acids in a protein. What are the four bases of genetic code? ACGT is an acronym for the four types of bases found in a DNA molecule: adenine (A), cytosine (C), guanine (G), and thymine (T) Is the genetic code universal? It is considered universal because humans, animals, plants and bacteria all have the exact same genetic code. All known organisms have the same four nucleotide bases (adenine, cytosine, guanine and thymine) but are different due to different arrangements of these nucleotide bases. What is the application of the genetic code? The genetic code is the set of rules used by living cells to translate information encoded within genetic material (DNA or RNA sequences of nucleotide triplets, or codons) into proteins. Why is genetic code important? Without the genetic code, cells would not be able to make the proteins that are necessary for life. The genetic code is also important in the study of genetics. By understanding how the genetic code works, scientists are able to manipulate it to produce desired traits in organisms. The genetic code uses what language? The genetic code uses a four-letter language known as nucleotides, specifically adenine (A), thymine (T), cytosine (C), and guanine (G). These nucleotides form the building blocks of DNA and RNA, and their sequence determines the genetic information that is passed on from one generation to the next. Why is it called genetic? The word genetic comes from the Greek word genetikos, which comes from the word “genesis” meaning - origin. It is used to refer to “origin code” on in another word “ origin of life code”. 63 | P r o f. D r. A s a l A z i z Lecture no.10: Title of the lecture: DNA Damage and repair mechanism Since human DNA contains basically three components (deoxy- pentose sugar, nitrogen base and phosphate group) where these components govern the growth and development processes in his body. Therefore, any change or damage in these components might lead to the impairment of the DNA and affect the life of the individual. Still, changes do occur in the DNA due to many factors which could be environmental or sometimes due to normal metabolic processes inside the cell. These changes could occur at a rate of 10,000 to 1000,000 molecular lesions per cell per day. DNA damage may be however, modifying the nucleotide sequence in a variety of ways, causing changes in its coding properties or normal function in transcription or replication which can ultimately lead to mutations and genomic instability. This could result in the development of a variety of cancers including colon, breast, and prostate. What are types of DNA damage? DNA bases can be damaged by: (1) oxidative processes, (2) alkylation of bases, (3) base loss caused by the hydrolysis of bases, (4) DNA crosslinking, (5) DNA strand breaks, including single and double stranded breaks. What is the best example of DNA damage? 64 | P r o f. D r. A s a l A z i z Perhaps the best-known example of the link between environmental-induced DNA damage and disease is that of skin cancer, which can be caused by excessive exposure to UV radiation in the form of sunlight (and, to a lesser degree, the tanning beds). What is the most common damage on DNA? One of the most common causes of damage to DNA is oxidative damage. Oxidative damage takes place when hydroxyl radicals are introduced into the cell. They're typically introduced via UV radiation from excess sunshine or other sources. However, the two major sources of DNA damage include: 1. DNA damage due to Endogenous sources such as: attack by reactive oxygen species (free radicals) produced from normal metabolic byproducts especially the process of oxidative deamination beside damage due to the replication errors. 2. DNA damage due to Exogenous sources by external agents such as: 1. ultraviolet [UV-A 200–400 nm] radiation from the sun 2. visible light energy (up to 670–700 nm) 3. other radiation frequencies, including x-rays and gamma rays 4. hydrolysis or thermal disruption 5. certain drugs (anticancer agents in clinical use); these include: a. alkylating agents (e.g., cyclophosphamide, cisplatin), b. antimetabolites (e.g., 5-fluorouracil), c. topoisomerase inhibitors (e.g., etoposide), and d. cytotoxic antibiotics (e.g., bleomycin) Moreover, Quinolones are a key group of antibiotics that interfere with DNA synthesis by inhibiting topoisomerase, most frequently topoisomerase II (DNA gyrase), an enzyme involved in DNA replication. Nowadays, quinolones are widely used for treating a variety of infections. Quinolones are broad-spectrum antibiotics that are active against both Gram-positive and Gram- negative bacteria, including mycobacteria, and anaerobes. It is particularly active against Gram-negative bacteria, including Salmonella, Shigella, Campylobacter, Neisseria, and Pseudomonas. 65 | P r o f. D r. A s a l A z i z Common fluoroquinolones include: Ciprofloxacin. Delafloxacin. Gemifloxacin (recently removed from markets) Levofloxacin. Moxifloxacin. Norfloxacin (discontinued in 2023) Ofloxacin. On the other hand, Ciprofloxacin and other quinolones at >20 μg/ml inhibit peripheral blood lymphocyte (PBL) cell growth by 30 to 35%, causing impaired cell cycle progression through the S phase. Cell cycle analysis thus, indicates DNA synthesis to be inhibited by fluoroquinolones at these concentrations. In addition, ciprofloxacin increases DNA cleavage by topoisomerase IV leading to an increase in DNA nicks. 6. human-made mutagenic chemicals, especially aromatic compounds that act as DNA intercalating agents 7. infection with certain viruses. 8. excessive exposure to water “many researches showed that; there is a large loss of DNA in human remains that have been immersed for 72 hours. Freshwater, swamp water, and saltwater all showed a large loss of DNA over the 72-hour period. Consequences of DNA Damage in human cells: - 66 | P r o f. D r. A s a l A z i z  DNA damage in the reproductive cells The damage in mammalian germ cells