DNA Replication: Process and Key Steps

• The process of copying DNA to ensure that genetic information is passed to new cells is termed DNA replication
• DNA replication occurs during the S phase of the cell cycle
• It follows a semi-conservative model
• Each new DNA molecule consists of one original (parental) and one newly synthesized strand
• This process is essential for cell growth, repair, and reproduction
• Mistakes in replication can cause mutations, leading to genetic disorders or cancer
• Proofreading mechanisms ensure high fidelity

Key Steps of DNA Replication

• Unwinding of DNA happens when Helicase breaks hydrogen bonds between base pairs (A-T and G-C)
• This separates the strands and forms a replication fork
• DNA polymerase III adds complementary nucleotides to the template strand to form new complementary strands
• Synthesis occurs in the 5' to 3' direction
• The leading strand is synthesized continuously in the same direction as the replication fork
• The lagging strand is synthesized in Okazaki fragments, which are later joined by DNA ligase
• DNA polymerase III proofreads and removes incorrect nucleotides, reducing mutation rates

Role of Different Enzymes in DNA Replication

• Helicase: Unwinds the DNA by breaking hydrogen bonds between bases
• DNA Polymerase III: Adds nucleotides to the new strand in a 5' to 3' direction
• DNA Polymerase I: Removes RNA primers and replaces them with DNA
• Primase: Synthesizes RNA primers needed to start replication
• DNA Ligase: Joins Okazaki fragments on the lagging strand
• Topoisomerase (Gyrase): Prevents supercoiling ahead of the replication fork

Semi-Conservative Replication

• After DNA replication, each new DNA molecule consists of one original (parental) strand from the previous DNA molecule, and one newly synthesized strand
• This model ensures accuracy in genetic inheritance and prevents drastic mutations in the genome

Leading vs. Lagging Strand Replication

• Leading Strand: Synthesized toward the replication fork, continuously, requires one RNA primer, DNA polymerase III moves continuously
• Lagging Strand: Synthesized away from the replication fork, discontinuously (Okazaki fragments), requires multiple RNA primers, DNA polymerase III stops and starts; DNA ligase joins Okazaki fragments.

DNA Proofreading

• DNA polymerase III proofreads during replication, detecting mismatched bases
• If an incorrect nucleotide is added, DNA polymerase removes it and inserts the correct one

Types of Mutations in DNA Replication

• Base Substitutions (Point Mutations): A single base is replaced by another
• Silent Mutation: No effect on amino acid sequence
• Missense Mutation: Changes one amino acid, possibly affecting protein function
• Nonsense Mutation: Introduces a premature stop codon, leading to a nonfunctional protein
• Frameshift Mutations (Insertions & Deletions): Addition or removal of a nucleotide; shifting the reading frame
• Huntington's disease mutation, an example of frameshift mutation, is caused by excessive trinucleotide (CAG) repeats.

PCR (Polymerase Chain Reaction)

• Technique used to amplify DNA in a laboratory It follows three main steps:
• Denaturation (95°C): DNA strands are separated by breaking hydrogen bonds
• Annealing (50–60°C): Short DNA primers bind to the target sequence
• Extension (72°C): Taq polymerase extends the DNA strand by adding nucleotides
• This cycle is repeated 30+ times, producing millions of copies of the DNA sequence

Gel Electrophoresis

• Used to separate DNA fragments
• DNA samples are loaded into a gel matrix (agarose gel)
• An electric current is applied; DNA is negatively charged and moves toward the positive electrode
• Smaller DNA fragments move faster and travel further, while larger fragments move slower
• The result is a banding pattern, used in DNA profiling

PCR vs. DNA Replication in Cells

• Feature: Enzyme Used, Location, Primers, Strand Separation, Direction
• PCR uses Taq Polymerase in a lab (in vitro), with synthetic DNA primers and heat for strand separation, synthesizing in the 5' to 3' direction
• DNA Replication uses DNA Polymerase III & I in the cell nucleus, uses RNA primers made by primase and helicase for strand separation, synthesizing leading (continuous) & lagging strand (discontinuous)

DNA Replication in Prokaryotes vs. Eukaryotes

• Prokaryotes: One origin per chromosome, fast replication speed, circular DNA, DNA Polymerase I and III, longer Okazaki fragments (~1000 bp)
• Eukaryotes: Multiple origins per chromosome, slower replication speed, linear chromosomes, multiple polymerases (α, δ, ε), and shorter Okazaki fragments (~100-200 bp)

Importance of DNA Replication

• Genetic Testing: Identifies mutations in inherited diseases
• Forensics: DNA profiling in crime scene investigations
• Gene Therapy: Correcting genetic disorders using edited DNA
• Cancer Research: Understanding how mutations lead to uncontrolled cell growth
• Biotechnology: Producing recombinant DNA for medicine, such as insulin production

Gene Mutation

• Gene mutation is a permanent change in the nucleotide sequence of DNA within a gene
• Mutations in germ-line cells (gametes) are heritable and can be passed to offspring
• Mutations in somatic (body) cells are not inherited but can lead to conditions such as cancer
• Mutations that occur in non-coding regions usually do not affect an organism unless they influence gene expression
• Mutations are the primary source of genetic variation, which is essential for evolution

Causes of Gene Mutations

• Mutations can occur randomly due to errors in DNA replication or repair
• They can also be caused by mutagenic agents, which increase the mutation rate above natural levels
• Types of Mutagens: Physical Mutagens, Chemical Mutagens, Biological Mutagens
• Physical Mutagens: UV radiation, X-rays; causes DNA strand breaks or thymine dimers, leading to errors in replication
• Chemical Mutagens: Reactive oxygen species (ROS), arsenic, alkylating agents; alters DNA bases, leading to incorrect base pairing
• Biological Mutagens: Viruses (HPV), bacteria (H. pylori), can insert viral DNA into the host genome, disrupting gene function

Types of Gene Mutations

• Point Mutations (Affecting a Single Base)
• Silent Mutation: A change in the DNA sequence that does not alter the amino acid sequence due to the redundancy of the genetic code
• Example: A mutation from GGA → GGU still codes for Glycine, so no functional change occurs
• Missense Mutation: A single nucleotide change results in a different amino acid, possibly altering protein function
• Example: Sickle Cell Anemia results from a mutation in the HBB gene (GAG → GUG), changing Glutamic Acid to Valine in hemoglobin
• Nonsense Mutation: A mutation changes a codon into a STOP codon, leading to an incomplete and often nonfunctional protein
• Example: Duchenne Muscular Dystrophy, where premature stop codons prevent the full formation of the dystrophin protein

Frameshift Mutations

• Occur when nucleotides are inserted or deleted, altering the reading frame of the genetic code
• Changes all codons after the mutation, leading to a completely different amino acid sequence
• Often results in a nonfunctional or truncated protein
• Example: Cystic Fibrosis can result from a 3-base deletion in the CFTR gene, leading to the loss of a single amino acid (Phe-508), which disrupts protein folding

Block Mutations

• Block mutations involve large-scale changes to chromosome structure, affecting multiple genes
• Duplication: A segment of the chromosome is copied, increasing gene expression, Charcot-Marie-Tooth disease
• Deletion: A segment is removed, leading to missing genes, Cri-du-chat syndrome
• Inversion: A chromosome segment is flipped, disrupting gene regulation, Some forms of hemophilia
• Translocation: A segment of one chromosome is moved to another, potentially disrupting genes, Chronic myeloid leukemia

Contribution of Mutations to Genetic Variation

• Mutations are the only way to create entirely new alleles in a population
• Some mutations provide beneficial adaptations driving evolution by natural selection
• Example: Sickle cell mutation (HbS allele) provides resistance to malaria, giving a survival advantage in regions where malaria is common

Germ-Line vs. Somatic Cell Mutations

• Germ-line mutation occurs in gametes (sperm or eggs)
• Effects of germ-line mutation is a passed to offspring, contributing to inherited genetic disorders
• Somatic mutation occurs in body cells (not gametes)
• Somatic mutations cannot be inherited but can lead to diseases like cancer

Mutations & Genetic Diseases

• Some mutations lead to genetic disorders due to defective proteins
• Examples of Genetic Diseases Caused by Mutations:
• Sickle Cell Anemia: Missense mutation in the HBB gene
• Cystic Fibrosis: Deletion mutation in the CFTR gene
• Huntington's Disease: Trinucleotide repeat expansion in the HTT gene
• Cancer: Mutations in tumor suppressor genes (p53, BRCA1)

Gene vs. Chromosomal Mutations

• Gene Mutations affect a single gene, altering protein function (e.g., sickle cell anemia, cystic fibrosis)
• Chromosomal Mutations involve large changes in chromosome structure or number (e.g., Down syndrome, Turner syndrome)

Mutations effect on Cancer

• Mutagens can alter DNA in ways that activate oncogenes or disable tumor suppressor genes, leading to uncontrolled cell division
• UV radiation can cause thymine dimers, which, if not repaired, can lead to skin cancer
• The HPV virus inserts viral DNA into host genes, increasing the risk of cervical cancer
• Preventative measures: Avoiding radiation, reducing exposure to carcinogens, and getting vaccines (e.g., HPV vaccine)

Translation

• Translation is the process of synthesizing proteins from mRNA, occurring in the ribosomes located in the cytoplasm or attached to the rough endoplasmic reticulum (RER) in eukaryotic cells
• It follows the central dogma of molecular biology, where genetic information flows from DNA → mRNA → Protein
• Translation ensures that the correct sequence of amino acids is assembled to form functional proteins

Stages of Translation

• Initiation: Small ribosomal subunit binds to the 5' end of the mRNA and moves along until it reaches the start codon (AUG), initiator tRNA (Methionine) binds to the start codon and the large ribosomal subunit attaches
• Elongation: Next tRNA enters the A-site of the ribosome and binds to the complementary mRNA codon, a peptide bond forms between the amino acids in the P-site and A-site and the ribosome shifts one codon forwards
• Termination: Translation continues until a stop codon (UAA, UAG, UGA) is reached, a release factor binds to the stop codon, triggering the disassembly of the ribosome and the release of the completed polypeptide

Key molecules involved in translation

• mRNA (messenger RNA): Carries the genetic code from DNA, specifying the amino acid sequence
• Ribosome (rRNA + proteins): Site of translation; facilitates codon-anticodon pairing and peptide bond formation
• tRNA (transfer RNA): Delivers amino acids to the ribosome by recognizing mRNA codons via its anticodon
• Amino acids: The building blocks of proteins, joined by peptide bonds
• Release factor: Recognizes stop codons and terminates translation

tRNA in Translation

• tRNA plays a crucial role in translation by delivering amino acids to the ribosome
• Each tRNA molecule has a specific anticodon that complementarily binds to an mRNA codon
• At the opposite end, tRNA carries the corresponding amino acid
• The aminoacyl-tRNA synthetase enzyme ensures that the correct amino acid is attached to the correct tRNA, a process called tRNA charging
• This specificity ensures the correct primary structure of the protein

Ribosome

• The ribosome has three key binding sites: A-site (holds the incoming tRNA), P-site (holds the growing polypeptide chain), E-site (where empty tRNA exits the ribosome)
• The ribosome moves along the mRNA in the 5' to 3' direction, ensuring the sequential addition of amino acids

Translation Regulation - Prokaryotic vs. Eukaryotic

• Prokaryotic Translation: Cytoplasm, No mRNA processing, 70S ribosome, direct binding to Shine-Dalgarno sequence, faster (~20 amino acids per second)
• Eukaryotic Translation: Cytoplasm/RER, mRNA undergoes capping, polyadenylation, and splicing, 80S ribosome, ribosome scans for the 5' cap and finds AUG, Slower (~1-3 amino acids per second)

Effect of Translation to Protein Structure and Function

• The sequence of amino acids (primary structure) determines
• Folding into secondary and tertiary structures (alpha-helices and beta-sheets)
• Protein stability and function through hydrogen bonds, disulfide bridges, and hydrophobic interactions
• Enzymatic activity, receptor binding, and structural integrity in the cell
• Incorrect translation can misfold proteins, leading to diseases like cystic fibrosis, Alzheimer's, and sickle cell anemia

Post-Translational Modification

• Once translation is complete, proteins may undergo post-translational modifications (PTMs) to become fully functional
• Phosphorylation: Adds phosphate groups (e.g., activating enzymes like kinases)
• Glycosylation: Adds sugar groups, crucial for cell signaling and membrane proteins
• Proteolysis: Cleaving the polypeptide into an active form (e.g., insulin activation)
• These modifications determine protein localization, function, and stability

Mutations and Diseases effect on translation

• Translation errors can cause misfolded proteins, leading to genetic diseases
• Missense Mutation: Incorrect amino acid substitution = Sickle Cell Anemia
• Nonsense Mutation: Early stop codon / truncated protein = Duchenne Muscular Dystrophy
• Frameshift Mutation: Disrupts reading frame = Cystic Fibrosis
• These errors affect protein function, leading to disorders ranging from enzyme deficiencies to neurodegenerative diseases

Antibiotics effect on bacterial translation

• Many antibiotics target bacterial ribosomes (70S) while sparing eukaryotic 80S ribosomes, making them effective treatments
• Tetracycline: Blocks tRNA binding to the A-site for Gram-positive and Gram-negative
• Chloramphenicol: Inhibits peptide bond formation for Broad-spectrum antibiotics
• Streptomycin: Binds to 30S subunit, causing misreading for Tuberculosis bacteria

Transcription

• Transcription is the process of copying genetic information from DNA to messenger RNA (mRNA)
• It occurs in the nucleus of eukaryotic cells and the cytoplasm of prokaryotic cells
• It allows genetic information to be carried from the DNA (nucleus) to the ribosomes for protein synthesis
• It ensures that only the required genes are expressed when needed
• Transcription is the first step of gene expression, converting the genetic code in DNA into a form that can be translated into proteins

Stages of Transcription

• Initiation
• RNA polymerase binds to the promoter region of the gene
• Transcription factors assist in positioning RNA polymerase correctly
• The DNA double helix unwinds, and hydrogen bonds between complementary base pairs break, exposing the template strand
• Elongation
• RNA polymerase moves along the template strand in the 3' to 5' direction, synthesizing a complementary mRNA strand in the 5' to 3' direction
• Complementary base pairing occurs: Adenine (A) → Uracil (U); Thymine (T) → Adenine (A); Cytosine (C) → Guanine (G); Guanine (G) → Cytosine (C)
• As the mRNA strand elongates, the DNA strands rewind behind RNA polymerase
• Termination
• When RNA polymerase reaches a termination sequence, transcription stops
• The mRNA strand detaches from the DNA template
• In eukaryotes, the pre-mRNA undergoes modifications before leaving the nucleus

Factors and Functions involved in transcription

• DNA (Template Strand): Provides the genetic code for mRNA synthesis
• RNA Polymerase: Unwinds DNA, adds RNA nucleotides, and synthesizes mRNA
• Promoter Region: A DNA sequence where RNA polymerase binds to start transcription
• Transcription Factors: Proteins that help RNA polymerase bind to the promoter
• mRNA (Messenger RNA): Carries the genetic code from the nucleus to ribosomes for translation
• Termination Sequence: Signals RNA polymerase to stop transcription

Modifications in Eukaryotic mRNA

• Eukaryotic mRNA undergoes processing before leaving the nucleus
• Capping (5' cap addition) is done at the 5' end of mRNA to protects from degradation and helps ribosomes recognize the start site
• Splicing removes Introns (non-coding regions), such that Exons are joined to form the final mRNA sequence
• This allows for alternative splicing
• Polyadenylation (Poly-A Tail Addition) takes place when a long sequence of adenine (A) nucleotides addition at the 3' end of mRNA, enhances mRNA stability and regulates its translation

Transcription in Prokaryotic vs Eukaryotic cells

• Location: Prokaryotic - Cytoplasm, Eukaryotic - Nucleus
• mRNA Processing: Prokaryotic - None, Eukaryotic - Capping, splicing, and polyadenylation

Transcription Regulation

• Transcription is a key control point in gene expression
• Cells regulate transcription through Transcription Factors
• Epigenetic Modifications : DNA methylation and histone modification can enhance or suppress transcription
• Environmental Stimuli: Certain genes are only transcribed in response to signals

Mutations effect on Transcription

• Mutations in transcription-related sequences can impact gene expression
• Promoter Mutation: Reduces RNA polymerase binding, lowering transcription rates, leading to β-thalassemia
• Splicing Mutation: Alters intron/exon removal, leading to defective proteins, leading to Spinal muscular atrophy (SMA)
• Frameshift Mutation: Changes the reading frame, affecting the entire protein, leading to Cystic fibrosis (CFTR gene deletion)

Transcription Research

• mRNA Vaccines: Such as Pfizer-BioNTech and Moderna COVID-19 vaccines, use synthetic mRNA to trigger an immune response
• Gene Therapy: Modifying transcription to treat genetic disorders like Duchenne Muscular Dystrophy
• Cancer Research: Understanding how transcription factors like p53 regulate cell division can help in cancer treatment
• Stem Cell Therapy: Reprogramming cells by activating or repressing transcription factors