Week 2 lecture 4. Biochemical Principles: Nucleic Acids, DNA Replication, Transcription, and Pharmacology

Overview and Fundamentals of Nucleic Acids

• Subcellular Storage of Genetic Information:

  • Nuclear Genome: The vast majority of cellular genetic information is stored within the cell nucleus in the form of deoxyribonucleic acid (DNA), which consists of long polymers of deoxyribonucleotides.

  • Mitochondrial Genome: A small, independent genome is separately maintained within the mitochondria.

  • Inheritance: Genetic information encoded in DNA is inherited from parents and transmitted to subsequent generations.

• Chemical Components of Nucleotides and Nucleosides:

  • Nucleotide Subunits: Each nucleotide monomer contains three distinct chemical functional groups:

  • A nitrogen-containing heterocyclic base.

  • A five-carbon sugar moiety (specifically deoxyribose in DNA or ribose in RNA).

  • A phosphate group.

  • Nucleoside Definition: A molecule comprising only the nitrogenous base and the sugar moiety, lacking the phosphate group, is termed a nucleoside.

• Nitrogenous Bases:

  • Purines: Characterized by a double-ring structure, making them larger molecules.

  • Adenine ($A$)

  • Guanine ($G$)

  • Pyrimidines: Characterized by a single-ring structure, making them smaller molecules.

  • Cytosine ($C$)

  • Thymine ($T$) (present in DNA)

  • Uracil ($U$) / Uridine (present in RNA)

• Acid-Base Properties of Nucleic Acids:

  • Basicity of Bases: Bases are defined as proton ($H^+$) acceptors. The nitrogen atoms within purine and pyrimidine rings possess lone pairs of electrons capable of forming coordinate covalent bonds with protons, conferring weak basic properties.

  • Masking of Basicity: In intact double-stranded nucleic acids, these basic properties are masked due to extensive hydrogen bonding between complementary base pairs.

  • Overall Acidity of DNA: Because of the dominant acidic nature of the phosphodiester sugar-phosphate backbone (due to ionized phosphate groups), DNA functions overall as a weak acid.

Nucleotide Nomenclature and Energetics

• Nomenclature Rules:

  • Structure Breakdown Example (Deoxyadenosine Monophosphate - $dAMP$):

  • Deoxy- indicates the presence of deoxyribose sugar (lacking an oxygen atom at the $C2'$ position).

  • -adenosine reflects the structural modification of the base adenine when covalently linked to the sugar.

  • -monophosphate indicates the attachment of a single phosphate group.

  • Base-to-Nucleoside Naming Transitions:

  • Adenine $\rightarrow$ Adenosine

  • Guanine $\rightarrow$ Guanosine

  • Thymine $\rightarrow$ Thymidine

  • Cytosine $\rightarrow$ Cytidine

• Energy-Carrying Nucleotides:

  • Nucleotide Triphosphates: Beyond storing genetic code, nucleotides function as universal energy carriers within cellular metabolism.

  • Adenosine Monophosphate ($AMP$): Contains one phosphate group.

  • Adenosine Diphosphate ($ADP$): Contains two phosphate groups.

  • Adenosine Triphosphate ($ATP$): Contains three phosphate groups; serves as the universal cellular energy currency.

  • Alternative energy carriers include Guanosine Triphosphate ($GTP$) and Cytidine Triphosphate ($CTP$).

• Thermodynamic Mechanism of Energy Storage and Release:

  • Electrostatic Repulsion: The adjacent phosphate groups in $ATP$ carry negative charges at physiological $pH$. This close proximity creates strong electrostatic repulsion.

  • Molecular Spring Analogy: Significant chemical energy is required during synthesis to overcome this repulsive force and hold the phosphate groups together, analogous to compressing a mechanical spring.

  • Phosphate Hydrolysis: Cleavage of phosphoanhydride bonds via hydrolysis is an exothermic reaction. The released energy/heat directly powers endergonic cellular work.

  • Energetics Quantified:

  • Hydrolysis of a single terminal phosphate group releases approximately $30\,\text{kJ}$ of energy.

  • Hydrolysis releasing a inorganic pyrophosphate moiety ($PP_i$, two bound phosphate groups) is followed immediately by enzymatic cleavage of $PP_i$ into two orthophosphates. This consecutive reaction yields a total cumulative energy release of $45.6\,\text{kJ}$.

• Biosynthetic Relationship Between Ribonucleotides and Deoxyribonucleotides:

  • Structural Difference: $ATP$ contains ribose sugar, whereas deoxyadenosine triphosphate ($dATP$) contains deoxyribose sugar, which lacks a hydroxyl ($-OH$) group at the $C2'$ carbon of the ring.

  • Unified Biosynthetic Pathway: Cells do not maintain separate parallel pathways for synthesizing ribonucleotides and deoxyribonucleotides. Instead, cells continuously synthesize ribonucleotides (e.g., $ATP$) and convert them into deoxyribonucleotides (e.g., $dATP$) as needed.

  • Temporal Demand: Ribonucleotides are consumed constantly for energy and transcription, whereas deoxyribonucleotides are required primarily during the $S$ phase of the cell cycle (DNA replication) or during DNA repair.

  • Enzymatic Reduction: The enzyme ribonucleotide reductase reduces the $C2'$ position of ribonucleotides (such as $ATP$) to yield corresponding deoxyribonucleotides (such as $dATP$).

  • Oncology Target: Ribonucleotide reductase is a key target for antineoplastic cancer therapies. Because malignant cells replicate rapidly and require heightened deoxyribonucleotide synthesis, inhibiting ribonucleotide reductase suppresses tumor growth.

Single-Stranded and Double-Stranded DNA Structure

• Cyclic Nucleotides as Second Messengers:

  • Cyclic AMP ($cAMP$): A primary intracellular messenger molecule.

  • Biosynthesis: Synthesized by the enzyme adenylate cyclase, which cleaves pyrophosphate from $ATP$ and forms a cyclic phosphodiester bond connecting the oxygen of the first phosphate group to the $C3'$ carbon of the ribose ring.

  • Signaling Mechanism: Hormones or neurotransmitters bind cell surface receptors to activate adenylate cyclase. The generated $cAMP$ binds downstream effector proteins, inducing conformational changes that either activate or deactivate them to initiate signaling cascades.

• Primary Structure of DNA:

  • Linear Polymerization: Nucleotides act as monomers covalently linked to form linear polynucleotide strands.

  • Sequence Definition: The specific linear arrangement of distinct nucleotides along a single strand constitutes the primary DNA structure (2D representation).

  • Phosphodiester Bonds: Monomers are joined by phosphodiester linkages, where a phosphate group forms two ester bonds: one to the $C3'$ hydroxyl of one sugar ring and the other to the $C5'$ hydroxyl of the adjacent sugar ring.

  • Directionality: Polynucleotide strands are strictly synthesized in a $5' \rightarrow 3'$ direction, meaning incoming nucleotide triphosphates are appended exclusively to the free $3'$ hydroxyl end of the growing chain.

• Secondary Structure and Double Helix Dynamics:

  • Three-Dimensional Helix: Secondary structure refers to the double-helical 3D conformation formed by two complementary polynucleotide strands coiling around a central axis.

  • Base Pairing Complementarity:

  • Adenine ($A$) pairs specifically with Thymine ($T$) via $2$ hydrogen bonds.

  • Guanine ($G$) pairs specifically with Cytosine ($C$) via $3$ hydrogen bonds.

  • Biological Consequence: Biological processes requiring physical strand separation (e.g., replication initiation) preferentially originate at $AT$-rich genomic regions due to the lower thermodynamic stability of $2$ hydrogen bonds compared to $3$ bonds in $GC$ pairs.

  • Stabilizing Intermolecular Forces:

  1. Hydrogen Bonding: Formed between complementary pairs ($A=T$ and $G \equiv C$).

  2. Hydrophobic Interactions and Base Stacking: Planar, hydrophobic nitrogenous bases are driven inward away from surrounding aqueous solvent, stacking vertically on top of one another to drive strand association.

  • Helical Geometry:

  • Uniform diameter: The overall distance between the two sugar-phosphate backbones is uniform across the entire helix, measuring approximately $2\,\text{nm}$. This consistency occurs because base pairing strictly pairs a double-ring purine with a single-ring pyrimidine.

  • Antiparallel Orientation: One strand runs $5' \rightarrow 3'$, while the complementary strand runs $3' \rightarrow 5'$.

  • Grooves and Hydration: The helical turn creates two distinct external grooves:

    • Major Groove: Wide and shallow.

    • Minor Groove: Narrow and deep.

    • Base edges remain exposed to solvent within these grooves. Water molecules form hydrogen bonds with these edges (hydration shell), facilitating sequence-specific interactions with DNA-binding proteins (typically targeting the major groove) and therapeutic drugs (targeting major or minor grooves to disrupt replication/transcription).

• Tertiary Conformation and Supercoiling:

  • Bacterial Genomes: Circular DNA molecules undergo localized twisting upon themselves in clockwise or counterclockwise directions, creating supercoiled tertiary structures that dictate genomic compaction and accessibility.

  • Eukaryotic Genomes: Linear chromosomes undergo localized twisting during active transcription or replication, acquiring contorted, compact structures known as hypercoiling.

  • Clinical Significance: Enzymes regulating supercoiling are critical targets for both antibacterial and chemotherapeutic agents.

DNA Replication Mechanics and Regulation

• Semiconservative Mechanism:

  • Prior to cell division, the double helix unwinds, and each parental strand serves as a template for the synthesis of a complementary daughter strand.

  • Each resulting double helix contains one original (parental) strand and one newly synthesized strand.

• Cell Cycle Stages and Replication Kinetics:

  • Mitosis ($M$ Phase): Cell division resulting in two daughter cells; duration is approximately $2\,\text{hours}$.

  • $G_1$ Phase: Post-mitotic growth phase characterized by intensive protein and lipid synthesis. Duration is highly variable, ranging from minutes to several months.

  • $G_0$ Phase: Quiescent state entered by non-dividing or terminally differentiated cells that arrest in $G_1$ indefinitely.

  • $S$ Phase (Synthesis Phase): Dedicated phase where nuclear DNA replication occurs. Once initiated, $S$ phase strictly completes within $9\text{--}12\,\text{hours}$.

  • Replication Foci: Newly synthesized DNA organizes into distinct nuclear foci, each measuring approximately $120\,\text{nm}$ in diameter and encompassing roughly $1,000,000\,\text{base pairs}$ ($1\,\text{Mb}$) of replicating DNA.

  • $G_2$ Phase: Post-synthetic phase dedicated to proofreading, DNA error repair, and mitotic preparation; duration is approximately $3\text{--}4\,\text{hours}$.

• Three Stages of DNA Replication:

  • 1. Initiation:

  • Soluble nuclear proteins identify and bind to specific $AT$-rich sequences designated as origins of replication.

  • Additional accessory factors assemble to form a multiprotein pre-replication complex at the conclusion of $G_1$ phase.

  • Upon entry into $S$ phase, the complex is enzymatically activated. The enzyme helicase unwinds the double helix by breaking hydrogen bonds at the $AT$-rich origin.

  • Two divergent helicases establish two opposing replication forks that move bidirectionally.

  • 2. Elongation:

  • DNA Polymerases: Catalyze phosphodiester bond formation.

    • Class I (DNA Polymerase I): Replaces RNA primers with deoxyribonucleotides and assists in replication repair.

    • Class II (DNA Polymerase II): Dedicated primarily to repairing replication errors.

    • Class III (DNA Polymerase III): Main workhorse enzyme responsible for primary elongation of leading and lagging strands.

  • Primer Requirement: DNA polymerases cannot initiate synthesis de novo on single-stranded DNA; they strictly require a double-stranded priming region with a free $3'-OH$ group.

  • Primase Activity: The enzyme primase binds single-stranded DNA and synthesizes a short complementary RNA primer.

  • Leading Strand Synthesis: Occurs continuously in the $5' \rightarrow 3'$ direction toward the advancing replication fork.

  • Lagging Strand Synthesis: Occurs discontinuously in the direction opposite fork movement. Primase repeatedly lays down RNA primers as new single-stranded template is exposed, and DNA Polymerase III synthesizes discrete segments termed Okazaki fragments.

  • 3. Termination:

  • Occurs when adjacent replication forks moving from neighboring origins converge.

  • Telomeric Termination: At linear chromosome ends (telomeres), DNA Polymerase I hydrolyzes the terminal RNA primers, replacing ribonucleotides with deoxyribonucleotides.

  • Ligation: The enzyme DNA ligase covalently seals nicked phosphodiester backbones between adjacent Okazaki fragments, creating a continuous strand.

  • Replication protein complexes disassemble, and intact daughter double helices re-coil.

• Cellular Regulation of Division Potential:

  • Non-Replicating (Terminally Differentiated) Cells: Permanently exit the cell cycle and never replicate DNA (e.g., mature neurons, skeletal muscle cells, cardiac myocytes, mature adipocytes, gametes).

  • Inducible Replication Cells: Quiescent cells that re-enter $S$ phase in response to specific physiological signals or injury (e.g., embryonic stem cells, adult stem cells, hepatocytes, basal skin keratinocytes).

  • Uncontrolled Replication: Pathological states characterized by loss of growth factor regulation (e.g., neoplastic cancer cells, host cells hijacked by viral infections).

Pharmacological Inhibitors of DNA Synthesis

• Chemotherapeutic Principles and Toxicity:

  • Therapeutic strategy: Selectively disrupt DNA replication to selectively eliminate rapidly dividing malignant cells or viral pathogens.

  • Clinical Side Effects: Non-specific inhibition of rapidly dividing host physiological tissues produces characteristic toxicities, impacting hematopoietic stem cells in bone marrow (myelosuppression), cutaneous epithelia, and hair follicle matrix cells (alopecia).

• Topoisomerase Inhibitors:

  • Topoisomers: Conformational isomers of DNA differing only in their topological supercoiling state.

  • Torsional Strain: Forward progression of the replication fork forces torsional unwinding strain ahead of the fork, forming tight supercoiled nodes that physical block further progression.

  • Enzymatic Function:

  • Type I Topoisomerase: Induces a transient single-strand break in DNA, passes the intact strand through the break to relieve torsional strain, and reseals the phosphodiester backbone.

  • Type II Topoisomerase: Induces transient double-strand breaks, rotates or passes double-stranded segments through, and reseals both backbones.

  • Antineoplastic Topoisomerase I Inhibitors:

  • Topotecan and Irinotecan (darenotecan).

  • Mechanism: These agents intercalate between adjacent DNA base pairs directly at the cleavage site formed by Topoisomerase I. They physically prevent the enzyme from resealing the broken strand, generating persistent single- and double-strand DNA breaks that trigger apoptotic cell death.

  • Antibacterial Topoisomerase Inhibitors:

  • Fluoroquinolones: Class of broad-spectrum antibiotics.

  • Mechanism: Selectively inhibit bacterial Type II topoisomerase, termed DNA gyrase.

  • Selectivity: Bacterial DNA gyrase possesses structural differences from human topoisomerases, permitting selective bacterial lethality without damaging human host tissues.

• Nucleotide Analogue Antimetabolites:

  • Gemcitabine:

  • Chemical structure: Analogous to deoxycytidine.

  • Mechanism: Direct incorporation into growing DNA chains during elongation in place of deoxycytidine.

  • Action: Functions as a obligate chain terminator; once incorporated, it physically blocks further addition of downstream nucleotides, permanently halting strand extension and inducing cell death.

  • Fluorouracil (5-FU / Chlorourocil):

  • Chemical structure: Analogous to pyrimidines (uridine and thymine).

  • Mechanism: Acts indirectly without direct incorporation into DNA polymer chains.

  • Target: Inactivates the enzyme thymidylate synthase (thymidylase synthase), which catalyzes the essential conversion of uridine monophosphate to thymidine monophosphate.

  • Action: Permanently inactivates thymidylate synthase, depleting intracellular thymidine pools required for DNA synthesis, thereby arresting replication.

RNA Synthesis, Structure, and Biological Roles

• Mechanisms of Transcription:

  • Transcription is the process of copying genomic DNA sequence information into a complementary single-stranded RNA molecule.

  • RNA Polymerase Dynamics:

  • Unwinds localized segments of double-stranded DNA.

  • Reads the DNA template strand exclusively in the $3' \rightarrow 5'$ direction.

  • Synthesizes single-stranded RNA in the antiparallel $5' \rightarrow 3'$ direction by adding matching ribonucleotide triphosphates to the free $3'-OH$ terminus.

  • Strand Terminology:

  • Template Strand: The DNA strand bound and read by RNA polymerase.

  • Coding Strand: The non-template DNA strand whose nucleotide sequence is identical to the synthesized RNA transcript (with Thymine replaced by Uracil).

• Chemical Differences Between RNA and DNA:

  • Sugar Moiety: RNA nucleotides contain ribose, characterized by a reactive hydroxyl ($-OH$) group at the $C2'$ carbon. DNA contains deoxyribose, possessing a single hydrogen ($-H$) at $C2'$.

  • Pyrimidines: RNA utilizes Uracil (Uridine) in place of Thymine.

  • Structural Variance: Thymine possesses a methyl ($-CH_3$) group on its ring, whereas Uracil lacks this methyl modification.

  • Base Pairing: Uracil forms $2$ hydrogen bonds with Adenine, identical to Thymine.

  • Intramolecular Folding: Although synthesized as single-stranded polymers, RNA molecules spontaneously bend and fold back on themselves, creating complex secondary and tertiary structures stabilized by localized internal hydrogen bonding.

• Biological Impact of Uracil in DNA:

  • Accidental incorporation of deoxyuridine into DNA triggers immediate cellular DNA damage response systems.

  • Failure to excise incorporated deoxyuridine increases somatic mutation rates.

  • Immune System Exploitation: Specialized immune cells intentionally substitute deoxythymidine with deoxyuridine within immunoglobulin genes. This controlled mutagenesis accelerates somatic hypermutation, greatly expanding antibody diversity.

• Biosynthetic Conversion of Uridine to Thymine:

  • Uridine acts as the metabolic precursor to Thymine. Cells utilize uridine ribonucleotides throughout the cell cycle and convert a fraction into thymine nucleotides during $S$ phase.

  • Two-Step Conversion Pathway:

  1. Uridine monophosphate is enzymatically reduced at the sugar ring to form deoxyuridine monophosphate ($dUMP$).

  2. $dUMP$ is methylated by thymidylate synthase to yield deoxythymidine monophosphate ($dTMP$).

• Functional Classes of Non-Coding and Coding RNA:

  • Messenger RNA ($mRNA$): Coding RNA; carries translated genetic codes from nuclear DNA to cytoplasmic ribosomes for polypeptide synthesis.

  • Ribosomal RNA ($rRNA$): Non-coding RNA; synthesized within the nucleolus. Acts as the enzymatic and structural backbone of ribosomal subunits.

  • Transfer RNA ($tRNA$): Non-coding RNA; functions as adaptor molecules transporting specific amino acids to ribosomes during translational protein synthesis.

  • Short Interfering RNA ($siRNA$): Small non-coding regulatory RNAs; bind complementary target $mRNA$ sequences to induce endonucleolytic degradation, selectively silencing gene expression.

Retroviral Replication and Antiviral Pharmacology

• Characteristics of Viral Genomes:

  • Eukaryotes universally utilize both DNA (storage) and RNA (expression). Viruses utilize either DNA or RNA exclusively as their genomic material.

  • Viral structures are simple, comprising a DNA or RNA genome encased within a protective protein capsid, relying entirely on host metabolic machinery for replication.

• Lifecycle of Retroviruses:

  • Viral Entry: Infection initiates when retroviral surface glycoproteins bind host membrane receptors, mediating viral entry via receptor-mediated endocytosis or direct envelope fusion.

  • Reverse Transcription: Retroviruses contain single-stranded RNA genomes and carry an encapsulated protein named reverse transcriptase.

  • Replication Cascade:

  1. Reverse transcriptase reads single-stranded viral RNA to synthesize a complementary single-stranded DNA copy.

  2. Reverse transcriptase synthesizes a second complementary strand, yielding double-stranded viral DNA.

  3. The viral DNA integrates permanently into the host cell nuclear genome.

  4. Host RNA polymerase transcribes integrated viral genes into viral $mRNA$ and genomic RNA.

  5. Host ribosomes translate viral structural and enzymatic proteins.

  6. New viral capsids assemble, encapsulate viral RNA, and bud from the host membrane.

• Human Immunodeficiency Virus (HIV) Pathology:

  • Target Cells: HIV infects host immune cells expressing surface receptors, specifically macrophages and $CD4^+$ T helper lymphocytes.

  • Clinical Progression: Progressive depletion of infected $CD4^+$ T cells results in Acquired Immunodeficiency Syndrome (AIDS).

  • Epidemiology: Over $1,000,000$ individuals in the United States and over $40,000,000$ individuals globally live with HIV infection.

• Antiviral Nucleoside Analogue Therapeutics:

  • Azidothymidine (AZT / Zidovudine):

  • Approved in $1987$ as the first effective antiretroviral therapy for HIV.

  • Chemical Structure: Nucleoside analogue of thymidine lacking a free $3'-OH$ group, possessing a $3'$-azido ($-N_3$) group instead.

  • Prodrug Activation: Must undergo intracellular phosphorylation by host kinases to convert into its active triphosphate form.

  • Mechanism: Competes directly with endogenous deoxythymidine for incorporation into synthesizing DNA strands.

  • Chain Termination: Because AZT lacks a $3'-OH$ group, reverse transcriptase cannot form the requisite phosphodiester bond with subsequent incoming nucleotides, completely terminating viral DNA synthesis.

  • Selective Toxicity: Active AZT-triphosphate exhibits approximately $100 \times$ higher binding affinity for viral reverse transcriptase compared to human nuclear DNA polymerases, minimizing severe host cytotoxic side effects.

  • Dideoxycytidine ($ddC$ / Zalcitabine):

  • Cytidine nucleoside analogue operating via an identical biochemical mechanism to AZT.

  • Lacks a $3'-OH$ group on its sugar ring, inducing chain termination upon incorporation by reverse transcriptase to halt retroviral replication.