Biochemical Genetics PDF

Biochemical genetics Definition: A branch of genetics at a biochemical level that in which the relationship of genes and their control over the function of an enzyme is observed. Biochemistry is the study of the chemical substances and vital processes occurring in live organisms. Bio...

Biochemical genetics Definition: A branch of genetics at a biochemical level that in which the relationship of genes and their control over the function of an enzyme is observed. Biochemistry is the study of the chemical substances and vital processes occurring in live organisms. Biochemists focus heavily on the role, function, and structure of biomolecules. The study of the chemistry behind biological processes and the synthesis of biologically active molecules are applications of biochemistry. Biochemistry studies life at the atomic and molecular level. Genetics is the study of the effect of genetic differences in organisms. This can often be inferred by the absence of a normal component (e.g. one gene). Molecular biology is the study of molecular underpinnings of the biological phenomena, focusing on molecular synthesis, modification, mechanisms and interactions. The central dogma of molecular biology, where genetic material is transcribed into RNA and then translated into protein, despite being oversimplified, still provides a good starting point for understanding the field.  Biochemical genetics is a combination of biochemistry and genetics. Biochemistry deals largely with the structure and function of cellular components, such as proteins, carbohydrates, lipids, nucleic acids, and other biomolecules, and of their functions and transformations during life processes. Genetics is a basically a study in heredity, particularly the mechanisms of hereditary transmission, and the variation of inherited characteristics among similar or related organisms. Some of the branches of genetics include biochemical genetics, cytogenetics, developmental genetics, genetic engineering, etc. Thus, biochemical genetics is a branch of genetics that deals with the chemical structure of the genes and with the mechanisms by which the genes control and regulate the structure and synthesis of proteins. It studies the relationship of genes and their control over the function of enzymes in biochemical pathways. It is genetics in terms of the chemical (biochemical) events involved, as in the manner in which DNA molecules replicate and control the synthesis of specific enzymes by the genetic code. Biochemical genetics = Inborn errors of metabolism (IEM) Inborn errors of metabolism (congenital metabolic diseases or inherited metabolic disorders) form a large class of genetic diseases involving congenital disorders of enzyme activities. The majority are due to defects of single genes that code for enzymes that facilitate conversion of various substances (substrates) into others (products). In most of the disorders, problems arise due to accumulation of substances which are toxic or interfere with normal function, or due to the effects of reduced ability to synthesize essential compounds. Inborn errors of metabolism were studied for the first time by British physician Archibald Garrod (1857–1936), in 1908. He is known for work that prefigured the "one gene–one enzyme" hypothesis, based on his studies on the nature and inheritance of alkaptonuria. His seminal text, Inborn Errors of Metabolism, was published in 1923. These metabolic disturbances, if left untreated, can result in a range of medical and developmental outcomes, from cognitive impairment, organ failure and even death. Many of the negative consequences of IEM can be mitigated by early detection and treatment which can include both drug and dietary intervention, utilising foods for special medical purposes (FSMP’s). Classification: Traditionally the inherited metabolic diseases were classified as disorders of carbohydrate metabolism, amino acid metabolism, organic acid metabolism, or lysosomal storage diseases.  Individually IEM’s are rare but are collectively more common (3.5 -5.9% of the worldwide population). Many IEM’s are screened for at birth via the Heel Prick Test (Newborn blood spot test). The newborn blood spot test helps to check if babies have any of 9 rare conditions, some of which can be life-threatening:  Cystic fibrosis (CF)  Sickle cell disease (SCD)  Congenital hypothyroidism (CHT)  Phenylketonuria (PKU)  Medium-chain acyl-CoA dehydrogenase deficiency (MCADD)  Maple syrup urine disease (MSUD)  Isovaleric acidaemia (IVA)  Glutaric aciduria type 1 (GA1)  Homocystinuria (HCU) A healthcare professional will prick the baby's heel using a device that has a tiny needle and collect a few drops of blood on a special card. The card is then sent away to be tested. The possible results of the newborn blood spot test are:  no signs of any condition found (no conditions suspected) – most babies will have this result.  carrier of a condition.  signs of a condition found (condition suspected). Diagnosis: Dozens of congenital metabolic diseases are now detectable by newborn screening tests, especially expanded testing using mass spectrometry.  Common screening tests used in the last sixty years:  Ferric chloride test (detects abnormal metabolites in urine)  Ninhydrin paper chromatography (detects abnormal amino acid patterns)  Guthrie test (detects excessive amounts of specific amino acids in blood).  Quantitative measurement of amino acids in plasma and urine  IEX-Ninhydrin post-column derivitization liquid ion chromatography (detects abnormal amino acid patterns and quantitative analysis)  Urine organic acid analysis by gas chromatography–mass spectrometry  Plasma acylcarnitine analysis by mass spectrometry  Urine purine and pyrimidine analysis by gas chromatography-mass spectrometry Specific diagnostic tests (or focused screening for a small set of disorders):  Tissue biopsy: liver, muscle, brain, bone marrow  Skin biopsy and fibroblast cultivation for specific enzyme testing  Specific DNA testing Signs and symptoms Symptoms of common inborn errors of metabolism include: Developmental delays. Weight loss. Growth challenges. Seizures. Poor appetite. Low energy (lethargic). Unusual odors of urine, sweat or breath. Abdominal pain. Cystic fibrosis (CF): Cystic fibrosis is a disease that causes thick, sticky mucus to build up in the lungs, digestive tract, and other areas of the body. It is one of the most common chronic lung diseases in children and young adults. It is a life-threatening disorder. It is caused by a defective gene that makes the body produce abnormally thick and sticky fluid, called mucus. This mucus builds up in the breathing passages of the lungs and in the pancreas. Genetics:  Cystic fibrosis is inherited in an autosomal recessive manner.  It is caused by the presence of mutations in both copies (alleles) of the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Cystic fibrosis is inherited in an autosomal recessive pattern Pathophysiology (biochemical): The CFTR gene regulates the transport of salts and water through cell membranes, providing instructions for creating a pathway that allows the passage of chloride ions. A mutation in the CFTR gene can impair the normal function of chloride channels, leading to abnormal transport of chloride ions and water, resulting in the formation of The CFTR protein is a channel protein that controls the flow of H2O and Cl− ions in and out of cells inside the lungs. When the thick and abnormal mucus. CFTR protein is working correctly, ions freely flow in and out of the cells. However, when the CFTR protein is malfunctioning, these ions cannot flow out of the cell due to a blocked channel. This causes cystic fibrosis, characterized by the buildup of thick mucus in the lungs.  Sickle cell disease (SCD): Sickle cell disease (SCD), also simply called sickle cell, is a group of hemoglobin-related blood disorders that are typically inherited. The most common type is known as sickle cell anemia. Sickle cell anemia results in an abnormality in the oxygen-carrying protein haemoglobin found in red blood cells. This leads to the red blood cells adopting an abnormal sickle-like shape under certain circumstances; with this shape, they are unable to deform as they pass through Normal blood cells next to a capillaries, causing blockages. sickle blood cell, coloured Problems in sickle cell disease typically begin around 5 scanning electron microscope image to 6 months of age.  Genetics: Hemoglobin is an oxygen-binding protein, found in erythrocytes, which transports oxygen from the lungs (or in the fetus, from the placenta) to the tissues. Each molecule of hemoglobin comprises 4 protein subunits, referred to as globins. Normally, humans have:-  hemoglobin F (Fetal hemoglobin, HbF), consisting of two alpha (α- globin) and two gamma (γ-globin) chains. This dominates during development of the fetus and until about 6 weeks of age. Afterwards, haemoglobin A dominates throughout life.  hemoglobin A1, (Adult hemoglobin, HbA) which consists of two alpha and two beta (β-globin) chains. This is the most common human hemoglobin tetramer, accounting for over 97% of the total red blood cell hemoglobin in normal adults.  hemoglobin A2, (HbA2) is a second form of adult hemoglobin and is composed of two alpha and two delta (δ-globin) chains. This hemoglobin typically makes up 1-3% of hemoglobin in adults.  β-globin is encoded by the HBB (hemoglobin subunit beta) gene on human chromosome 11; mutations in this gene produce variants of the protein which are implicated with abnormal hemoglobins. The mutation which causes sickle cell disease results in an abnormal hemoglobin known as hemoglobin S (HbS), which replaces HbA in adults. Base-pair substitution that Sickle cell disease is inherited in causes sickle cell anemia an autosomal recessive pattern Pathophysiology: Under conditions of low oxygen concentration, HBS polymerises to form long strands within the red blood cell (RBC). These strands distort the shape of the cell and after a few seconds cause it to adopt an abnormal, inflexible sickle-like shape. This process reverses when oxygen concentration is raised and the cells resume their normal biconcave disc shape. Scanning electron micrograph showing a If sickling takes place in the venous system, after blood mixture of red blood cells, some with round has passed through the capillaries, it has no effect on the normal morphology, some with mild sickling organs and the RBCs can unsickle when they become showing elongation and bending oxygenated in the lungs. Repeated switching between sickle and normal shapes damages the membrane of the RBC so that it eventually becomes permanently sickled Congenital hypothyroidism (CHT): It is thyroid hormone deficiency present at birth. If untreated for several months after birth, severe congenital hypothyroidism can lead to growth failure and permanent intellectual disability. Significant deficiency may cause excessive sleeping, reduced interest in nursing, poor muscle tone, low or hoarse cry, infrequent bowel movements, significant jaundice, and low body temperature. Treatment consists of a daily dose of thyroid hormone (thyroxine) by mouth. 6 week old female with Genetics: jaundice due to hypothyroidism  Congenital hypothyroidism can also occur due to genetic defects of thyroxine or triiodothyronine synthesis within a structurally normal gland.  The condition usually has an autosomal recessive inheritance pattern.  When congenital hypothyroidism results from mutations in the (Paired box gene 8) PAX8 gene or from certain mutations in the (thyroid stimulating hormone receptor) TSHR or (Dual oxidase 2 ) DUOX2 gene, the condition has an autosomal dominant pattern of inheritance, which means one copy of the altered gene in each cell is sufficient to cause the disorder.  Phenylketonuria (PKU): It is an inborn error of metabolism that results in decreased metabolism of the amino acid phenylalanine. Untreated PKU can lead to intellectual disability, seizures, behavioral problems, and mental disorders. It may also result in a musty smell and lighter skin. It is caused by mutations in the (Phenylalanine hydroxylase) PAH gene, which can result in inefficient or nonfunctional phenylalanine hydroxylase (enzyme). This enzyme is necessary to metabolize the amino acid phenylalanine (Phe) to the amino acid tyrosine (Tyr). When PAH gene activity is reduced, phenylalanine accumulates and is converted into phenylpyruvate (also known as phenylketone), which can be detected in the urine This results in the buildup of dietary phenylalanine to potentially toxic levels. Genetics: It is autosomal recessive, meaning that both copies of the gene must be mutated for the condition to develop. Abnormally small head (microcephaly) Pathophysiology: complications of PKU include severe intellectual disability, brain function abnormalities, microcephaly, mood disorders, irregular motor functioning, and behavioral problems such as attention deficit hyperactivity disorder, as well as physical symptoms such as a "musty" odor, eczema, and unusually light skin and hair coloration. Medium-chain acyl-CoA dehydrogenase deficiency (MCADD): It is a disorder of fatty acid oxidation that impairs the body's ability to break down medium-chain fatty acids into acetyl- CoA. The disorder is characterized by hypoglycemia and sudden death without timely intervention, most often brought on by periods of fasting or vomiting. Prior to expanded newborn screening, MCADD was an underdiagnosed cause of sudden death in infants. Individuals who have been identified prior to the onset of symptoms have an excellent prognosis. Genetics:  MCADD is inherited in an autosomal recessive manner, meaning an affected individual must inherit a mutated allele from both of their parents.  Acyl-Coenzyme A dehydrogenase medium chain (ACADM) is the gene involved, located at 1p31, with 12 exons and coding for a protein of 421 amino acids.  There is a common mutation, replacement of an adenine at position 985 with guanine, which results in a substitution of lysine with glutamic acid at position 304 of the protein. Maple syrup urine disease (MSUD): It is a rare, inherited metabolic disorder that affects the body's ability to metabolize amino acids due to a deficiency in the activity of the branched- chain alpha-ketoacid dehydrogenase (BCKAD) complex. It particularly affects the metabolism of amino acids—leucine, isoleucine, and valine. With MSUD, the body is not able to properly break down these amino acids, therefore leading to the amino acids to build up in urine and become toxic. The condition gets its name from the distinctive sweet odor of affected infants' urine and earwax due to the buildup of these amino acids. Genetics:  Mutations in the following genes cause maple syrup urine disease:  BCKDHA (OMIM: 608348)  BCKDHB (OMIM: 248611)  DBT (OMIM: 248610)  DLD (OMIM: 238331) These four genes produce proteins that work together as the branched- chain alpha-keto acid dehydrogenase complex. Mutation in any of these genes reduces or eliminates the function of the enzyme complex, preventing the normal breakdown of isoleucine, leucine, and valine. It is an autosomal recessive inheritance pattern. Isovaleric acidemia (IVA): Isovaleric acidemia is a rare autosomal recessive metabolic disorder which disrupts or prevents normal metabolism of the branched-chain amino acid leucine. It is a classical type of organic acidemia. A characteristic feature of isovaleric acidemia is a distinctive odor of sweaty feet. This odor is caused by the buildup of a compound called isovaleric acid in affected individuals. The enzyme encoded by IVD, isovaleric acid-CoA dehydrogenase (EC 1.3.99.10), plays an essential role in breaking down proteins from the diet. Specifically, the enzyme is responsible for the third step in processing leucine, an essential amino acid. If a mutation in the IVD gene reduces or eliminates the activity of this enzyme, the body is unable to break down leucine properly. As a result, isovaleric acid and related compounds build up to toxic levels, damaging the brain and nervous system Glutaric aciduria type 1 (GA1): Glutaric acidemia type 1 (GA1) is an inherited disorder in which the body is unable to completely break down the amino acids lysine, hydroxylysine and tryptophan. Excessive levels of their intermediate breakdown products (glutaric acid, glutaryl- CoA, 3-hydroxyglutaric acid, glutaconic acid) can accumulate and cause damage to the brain (and also other organs), but particularly the basal ganglia, which are regions that help regulate movement. The condition is inherited in an autosomal recessive pattern: mutated copies of the gene glutaryl-CoA dehydrogenase (GCDH) must be provided by both parents to cause GA1. The GCDH gene encodes the enzyme glutaryl-CoA dehydrogenase. This enzyme is involved in degrading the amino acids lysine, hydroxylysine and tryptophan. Mutations in the GCDH gene prevent production of the enzyme or result in the production of a defective enzyme with very low residual activity. Homocystinuria (HCU): Homocystinuria (HCU) is an inherited disorder of the metabolism of the amino acid methionine due to a deficiency of cystathionine beta synthase or methionine synthase. It is an inherited autosomal recessive trait, which means a child needs to inherit a copy of the defective gene from both parents to be affected. Symptoms of homocystinuria can also be caused by a deficiency of vitamins B6, B12, or folate. This defect leads to a multi-systemic disorder of the connective tissue, muscles, central nervous system (CNS), and cardiovascular system. Cell cycle: Actively dividing eukaryote cells pass through a series of stages known collectively as the cell cycle: two gap phases (G1 and G2); an S (for synthesis) phase, in which the genetic material is duplicated; and an M phase, in which mitosis partitions the genetic material and the cell divides. G1 phase. Metabolic changes prepare the cell for division. At a certain point - the restriction point - the cell is committed to division and moves into the S phase. S phase. DNA synthesis replicates the genetic material. Each chromosome now consists of two sister chromatids. G2 phase. Metabolic changes assemble the cytoplasmic materials necessary for mitosis and cytokinesis. M phase. A nuclear division (mitosis) followed by a cell division (cytokinesis). The period between mitotic divisions - that is, G1, S and G2 - is known as interphase.  DNA replication: DNA replication, like all biological polymerization processes, proceeds in three enzymatically catalyzed and coordinated steps: initiation, elongation and termination. Sequences used by initiator proteins tend to be "AT-rich" (rich in adenine and thymine bases), because A-T base pairs have two hydrogen bonds (rather than the three formed in a C-G pair) and thus are easier to strand-separate. The eukaryotic pre-RC is the most complex and highly regulated pre-RC. In most eukaryotes it is composed A pre-replication complex (pre-RC) is a protein of six ORC proteins (ORC1-6), Cdc6 (cell division complex that forms at the origin of cycle 6), Cdt1 (Chromatin licensing and DNA replication replication during the initiation step of DNA factor 1) , and a heterohexamer of the six MCM replication. (minichromosome maintenance protein complex) proteins (MCM2-7). Sources/Agents of DNA Damage 1. Endogenous DNA Damage: Endogenous DNA damage originates from internal reactions involving chemically active DNA within cells. Replication errors are one source of endogenous DNA damage that occurs during DNA replication when incorrect nucleotides are inserted opposite the template bases. During replication, some DNA polymerases with lower fidelity can be involved, leading to potential errors. Topoisomerase enzymes are another source of endogenous DNA damage. Topoisomerases remove the supercoiling of DNA during replication and transcription. However, misalignment of the DNA ends can stabilize the topoisomerase-DNA cleavage complex and result in the formation of DNA lesions. Reactive oxygen species (ROS) are produced during cellular processes and can cause oxidative damage to DNA. While ROS plays an important role in normal cellular functions, excessive levels can lead to various DNA lesions and modifications. Excessive ROS has been associated with the development of several human diseases like cancer, Alzheimer’s disease, and diabetes. Alkylating agents are reactive compounds that can add methyl or ethyl groups to DNA bases, leading to chemical modifications. Spontaneous methylation events can generate different methylated bases. Some methylated bases are mutagenic and can lead to specific types of mutations. 2. Exogenous DNA Damage: Exogenous DNA damage is caused by external factors, such as environmental agents, physical forces, or chemicals. Ionizing radiation (IR) directly damages DNA or indirectly affects it through the generation of highly reactive hydroxyl radicals ( OH) from water molecules. IR can cause different types of damage to the DNA such as base lesions, and single-strand and double-strand breaks. Ultraviolet (UV) radiation is another agent of DNA damage. It is the leading cause of skin cancers. UV light can form pyrimidine dimers where two pyrimidines on the same DNA strand are joined together. This alteration in DNA structure can block transcription and replication processes. Exogenous alkylating agents, found in sources like tobacco smoke and industrial activities, react with DNA and can cause mutagenic and carcinogenic changes. They primarily target the nitrogenous bases in DNA. Examples of alkylating agents include sulfur and nitrogen mustards. Aromatic amines, found in cigarette smoke, fuel, coal, dyes, and pesticides, are also exogenous sources of DNA damage. These agents can create long- lasting lesions in the DNA structure that lead to the substitution of DNA bases and frameshift mutations. Polycyclic aromatic hydrocarbons (PAHs) are known carcinogens found in sources like tobacco smoke, automobile exhaust, and other environmental pollutants. PAHs require activation by the liver’s P-450 system to produce reactive substances that can potentially damage DNA. DNA damage: Types of DNA Damage and Mechanisms: 1. DNA Strand Breaks DNA strand breaks occur when one or both strands of DNA are interrupted. There are two types: single-strand breaks (SSBs) where one strand is cut, and double-strand breaks (DSBs) where both strands are cut. These breaks can be caused by ionizing radiation like X-rays and gamma rays, as well as certain chemicals. 2. Oxidative Damage Oxidative damage can occur due to the action of reactive oxygen species (ROS) which leads to the formation of lesions. The highly reactive ROS, such as hydroxyl radicals ( OH), can cause oxidative damage to DNA bases. 3. Alkylation of Bases Alkylating agents, both endogenous and exogenous, can modify DNA bases by introducing alkyl groups. These modifications can be cytotoxic, mutagenic, or have neutral effects on the cell. 4. Base Loss Base loss occurs when the nitrogenous bases in DNA are removed, leaving behind apurinic/apyrimidinic (AP) sites or abasic sites. AP sites are chemically unstable and can lead to DNA strand breaks or mutagenic events if left unrepaired. 5. Bulky Adduct Formation Bulky adducts are formed when certain chemicals, such as polycyclic aromatic hydrocarbons (PAHs), covalently bind to DNA bases. These adducts create bulky modifications that stick out from the DNA and disrupt its structure. They can interfere with DNA replication, transcription, and repair processes, potentially leading to mutations. 6. DNA Crosslinking DNA crosslinking occurs when two nucleotides in DNA become covalently linked together. Crosslinks can form within the same DNA strand (intrastrand crosslinks) or between opposite DNA strands (interstrand crosslinks). DNA crosslinks prevent the separation of DNA strands during replication or transcription, leading to the disruption of important cellular processes. DNA repair: DNA repair is a collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome. The rate of DNA repair depends on various factors, including the cell type, the age of the cell, and the extracellular environment. A cell that has accumulated a large amount of DNA damage or can no longer effectively repair its DNA may enter one of three possible states: an irreversible state of dormancy, known as senescence cell suicide, also known as apoptosis or programmed cell death unregulated cell division, which can lead to the formation of a tumor that is cancerous The DNA repair ability of a cell is vital to the integrity of its genome and thus to the normal functionality of that organism. Many genes that were initially shown to influence life span have turned out to be involved in DNA damage repair and protection. DNA Repair Types and Mechanisms 1. Direct reversal repair: Direct reversal repair is a DNA repair mechanism that directly fixes specific types of DNA damage without the need for excision or replacement. Two examples of DNA damage that can be reversed are UV- induced lesions and alkylated bases. UV-induced lesions, caused by UV light, can be reversed through a process called photoreactivation, which uses visible light energy to break the damaged DNA structure, restoring the original pyrimidine bases. Photolyases are found in bacteria, fungi, plants, and many vertebrates, but are not found in placental mammals. Alkylated bases can be reversed by enzymes such as O6- alkylguanine-DNA alkyltransferase (AGT) and AlkB-related dioxygenases, which removes or modifies the alkyl group, respectively. 2. Base excision repair Base excision repair (BER) is a DNA repair mechanism that removes and replaces damaged bases. It involves the action of various DNA glycosylases such as 8-oxoguanine DNA glycosylase (OGG1). These enzymes recognize and remove damaged bases. BER includes both short patch repair, where an abasic site is processed and filled by specific enzymes, and long patch repair, where gaps are tailored and DNA synthesis occurs followed by ligation. One example of BER is the repair of uracil-containing DNA. In this process, a DNA glycosylase recognizes and removes the uracil base, creating a gap in the DNA called AP site. The gap is then cleaved by an enzyme called AP endonuclease. After that, the remaining sugar is removed, and the gap is filled using DNA polymerase and sealed with ligase. 3. Nucleotide excision repair Nucleotide excision repair (NER) deals with bulky adducts and cross-linking lesions caused by UV radiation or chemical exposure. NER removes a fragment of nucleotides containing the damaged lesion and synthesizes a new DNA strand using the undamaged strand as a template. NER consists of two pathways: Global Genome NER (GG-NER) repairs bulky damages throughout the entire genome, including regions that are not actively transcribed. Transcription-Coupled NER (TC-NER repairs damage that occurs on the transcribed DNA strand. Mutations in NER pathway genes can lead to disorders such as xeroderma pigmentosum (XP) and certain other neurodegenerative conditions. Xeroderma pigmentosum (XP) is a genetic disorder in which there is a decreased ability to repair DNA damage such as that caused by ultraviolet (UV) light. 4. Mismatch repair Mismatch repair (MMR) pathway repairs base mismatches and insertion-deletion loops that occur during replication. Most of these errors are fixed by the proofreading activity of DNA polymerase during replication, but some may be missed and need to be corrected later. The MMR pathway involves three steps: recognition of mismatches, degradation of the error-containing strand, and synthesis of the correct DNA sequence. First, protein complexes such as (MutS homolog 2) MSH2-MSH6 in the MutS protein locate the mismatch errors and forms a complex with MutL which helps in further repair. MutS and MutL are important protein complexes in eukaryotes. In E. coli, another protein MutH also has an important role in mismatch repair. Next, exonuclease 1 (Exo1) degrades the error-containing strand while replication protein A (RPA) prevents further DNA degradation by binding to the exposed DNA. Then, DNA polymerase δ synthesizes the correct sequence. Finally, DNA ligase then seals any remaining nicks in the repaired DNA. Mutations in MMR genes can lead to Lynch syndrome, a hereditary condition associated with an increased risk of colon, ovarian, and other cancers. Lynch syndrome used to be called hereditary nonpolyposis colorectal cancer (HNPCC) 5. Single-strand break repair (SSBR) Single-stranded breaks (SSBs) in DNA can occur due to oxidative damage, abasic sites, or errors in the activity of the DNA topoisomerase enzyme. These breaks can disrupt DNA replication, halt transcription, and activate cellular processes that can lead to cell death. To protect the exposed single strand from breaking, (Poly [ADP-ribose] polymerase 1) PARP1 proteins coat the single strand and act as a shield. SSBR can be accomplished through various pathways already explained above, including base excision repair, nucleotide excision repair, and mismatch repair. 6. Double-strand break repair Double-strand breaks (DSBs) in DNA can be repaired through two pathways: homologous recombination (HR) and non-homologous end joining (NHEJ). Homologous recombination (HR) HR is a precise repair pathway that requires a matching DNA sequence as a template. It primarily uses the sister chromatid. HR is most active during the S, G2, and M phases of the cell cycle when sister chromatids are present. The HR process involves creating single-stranded DNA (ssDNA) by degrading one strand of the DNA break and coating it with proteins. Non-homologous end joining (NHEJ) NHEJ is a simple and widely used mechanism that directly seals the broken ends of DNA without the need for a homologous DNA template. It can occur throughout the cell cycle. The NHEJ pathway is faster but can be more error- prone compared to HR.  Protein synthesis It is a core biological process, occurring inside cells, balancing the loss of cellular proteins (via degradation or export) through the production of new proteins. Protein synthesis is a very similar process for both prokaryotes and eukaryotes but there are some distinct differences. Protein synthesis can be divided broadly into two phases - transcription and translation. Prof. Alyaa Farid Transcription Initially, an enzyme known as a helicase acts on the molecule of DNA. DNA has an antiparallel, double helix structure composed of two, complementary polynucleotide strands, held together by hydrogen bonds between the base pairs. The helicase disrupts the hydrogen bonds causing a region of DNA (corresponding to a gene) to unwind, separating the two DNA strands and exposing a series of bases. Despite DNA being a double stranded molecule, only one of the strands acts as a template for pre-mRNA synthesis - this strand is known as the template strand. The other DNA strand (which is complementary to the template strand) is known as the coding strand. The enzyme RNA polymerase binds to the exposed template strand and reads from the gene in the 3' to 5' direction. RNA polymerase binds to a sequence of DNA called the promoter, found near the beginning of a gene. Simultaneously, the RNA polymerase synthesizes a single strand of pre- mRNA in the 5'-to-3' direction by catalysing the formation of phosphodiester bonds between activated nucleotides (free in the nucleus) that are capable of complementary base pairing with the template strand. Despite the fast rate of synthesis, the RNA polymerase enzyme contains its own proofreading mechanism. The proofreading mechanisms allows the RNA polymerase to remove incorrect nucleotides (which are not complementary to the template strand of DNA) from the growing pre-mRNA molecule through an excision reaction. When RNA polymerases reaches a specific DNA sequence (terminator) which terminates transcription, RNA polymerase detaches and pre-mRNA synthesis is complete. Prof. Alyaa Farid  Translation During translation, ribosomes synthesize polypeptide chains from mRNA template molecules. In eukaryotes, translation occurs in the cytoplasm of the cell, where the ribosomes are located either free floating or attached to the endoplasmic reticulum. In prokaryotes, which lack a nucleus, the processes of both transcription and translation occur in the cytoplasm. Ribosomes are complex molecular machines, made of a mixture of protein and ribosomal RNA, arranged into two subunits (a large and a small subunit), which surround the mRNA molecule. The ribosome reads the mRNA molecule in a 5'-3' direction and uses it as a template to determine the order of amino acids in the polypeptide chain. In order to translate the mRNA molecule, the ribosome uses small molecules, known as transfer RNAs (tRNA), to deliver the correct amino acids to the ribosome. Each tRNA is composed of 70-80 nucleotides and adopts a characteristic cloverleaf structure due to the formation of hydrogen bonds between the nucleotides within the molecule. There are around 60 different types of tRNAs, each tRNA binds to a specific sequence of three nucleotides (known as a codon) within the mRNA molecule and delivers a specific amino acid. Prof. Alyaa Farid