Meditaliano IMAT Prep

Part 1: DNA Replication, Repair, and the Cell Cycle

This section details the high-fidelity process of DNA duplication, the surveillance systems that guard the integrity of the genome, and the regulatory network that governs cell division.

1.1 The Replication Machinery

• Initiation: Replication begins at specific sites called origins of replication. Prokaryotes have a single origin (OriC), while eukaryotes have multiple origins on each chromosome, allowing for rapid replication of their much larger genomes. The origin is recognized by initiator proteins (like ORC in eukaryotes), which recruit other proteins to form the pre-replication complex.
• Elongation - Key Enzymes:
  • Helicase: An ATP-dependent motor protein that unwinds the DNA double helix.
  • Single-Strand Binding (SSB) Proteins: Prevent re-annealing.
  • Topoisomerases: Relieve supercoiling. Type I enzymes make single-strand cuts, while Type II (like DNA gyrase in bacteria) make double-strand cuts. Cancer drugs like etoposide target human topoisomerase II.
  • Primase: Synthesizes a short RNA primer (an RNA polymerase).
  • DNA Polymerase: Synthesizes DNA 5'→3'. The leading strand is continuous; the lagging strand is discontinuous (Okazaki fragments). Eukaryotic DNA Pol α works with primase, Pol δ synthesizes the lagging strand, and Pol ε synthesizes the leading strand.
  • PCNA (Proliferating Cell Nuclear Antigen): A sliding clamp that encircles the DNA and tethers the polymerase to the template, dramatically increasing processivity.
  • DNA Ligase: Joins Okazaki fragments by forming a phosphodiester bond.
• Proofreading and Fidelity: The intrinsic 3'→5' exonuclease activity of replicative DNA polymerases provides a crucial proofreading function, dramatically increasing the fidelity of replication.

1.1.1 The End-Replication Problem and Telomerase

Linear eukaryotic chromosomes face a problem: the lagging strand cannot be fully replicated at its very end. This is because the final RNA primer, once removed, leaves a gap that DNA polymerase cannot fill (as it requires a 3'-OH group to build upon). This leads to progressive shortening of the chromosome with each cell division, known as the end-replication problem. To solve this, specialized structures called telomeres (repetitive DNA sequences, TTAGGG in humans) cap the ends. The enzyme telomerase, a ribonucleoprotein with its own internal RNA template, extends these telomeres. Most somatic cells have low telomerase activity, linking telomere shortening to cellular aging. Conversely, cancer cells often reactivate telomerase, enabling their immortality.

1.1.2 Key DNA Polymerases (Eukaryotic vs. Prokaryotic)

1.2 DNA Damage and Repair

1.2.1 In-Depth Look: Base Excision Repair (BER)

BER is a "cut-and-patch" mechanism for small, non-helix-distorting lesions. The process is precise:

1. Recognition & Excision: A specific DNA glycosylase scans the DNA, finds the damaged base (e.g., uracil formed from cytosine deamination), and flips it out. It then cleaves the N-glycosidic bond, removing the base and leaving an "abasic" or AP site (apurinic/apyrimidinic).
2. Incision: An AP endonuclease recognizes the AP site and cuts the phosphodiester backbone just 5' to the site.
3. Synthesis & Ligation: DNA Polymerase β (in humans) adds the correct nucleotide. Finally, DNA ligase seals the nick.

1.3 The Cell Cycle and Its Regulation

The cell cycle is driven by cyclin-dependent kinases (CDKs), activated by binding to cyclins. Key pairs include: Cyclin D/CDK4/6 (G1 progression), Cyclin E/CDK2 (G1/S transition), Cyclin A/CDK2 (S phase), and Cyclin B/CDK1 (M phase). Checkpoints ensure fidelity: the G1 checkpoint (the "restriction point") is controlled by the tumor suppressor Rb (Retinoblastoma), which inhibits E2F transcription factors. When Rb is phosphorylated by CDK4/6, it releases E2F, allowing transcription of S-phase genes. The G2/M checkpoint ensures DNA is fully replicated. The spindle checkpoint ensures all chromosomes are properly attached to the mitotic spindle before anaphase. The tumor suppressor p53 ("the guardian of the genome") can halt the cell cycle (by inducing a CDK inhibitor, p21) or trigger apoptosis in response to DNA damage.

1.3.1 Key Checkpoint Regulators (ATM/ATR)

At the heart of the DNA damage response are two master kinases: ATM and ATR. ATM is primarily activated by double-strand breaks, while ATR is activated by single-strand breaks or stalled replication forks. Once activated, they phosphorylate a host of downstream targets, including p53 and Chk1/Chk2 kinases, to coordinate cell cycle arrest, DNA repair, and (if necessary) apoptosis.

1.4 Mitosis vs. Meiosis and Apoptosis

Mitosis produces two genetically identical diploid (2n) daughter cells. Meiosis produces four genetically unique haploid (n) gametes through two rounds of division. Meiosis I is the reductional division where homologous chromosomes separate. A key event is crossing over (homologous recombination) during Prophase I, which creates genetic diversity. Meiosis II is an equational division where sister chromatids separate, similar to mitosis.

1.4.1 Mitosis vs. Meiosis: A Detailed Comparison

1.4.2 Apoptosis: Programmed Cell Death

Apoptosis is an orderly process essential for development and tissue homeostasis. The intrinsic (mitochondrial) pathway is controlled by the Bcl-2 family of proteins, leading to the release of cytochrome c. The extrinsic (death receptor) pathway is initiated by external signals binding to receptors like Fas. Both pathways converge on the activation of a cascade of proteases called caspases. Initiator caspases (like Caspase-8, 9) activate executioner caspases (like Caspase-3), which execute the dismantling of the cell by cleaving key proteins.

Part 2: Gene Expression and Regulation

This section covers the central dogma of molecular biology: the flow of genetic information from DNA to RNA to protein, and the complex layers of its regulation.

2.1 From Gene to Protein: Transcription and Translation

• Eukaryotic Transcription: Requires general transcription factors (e.g., TBP binding to the TATA box) to recruit RNA Polymerase II to the promoter. Gene-specific expression is controlled by specific transcription factors that bind to distant enhancers or silencers.
• Post-Transcriptional mRNA Processing: Involves 5' capping (a 7-methylguanosine cap), 3' polyadenylation (a poly-A tail), and splicing of introns by the spliceosome (a complex of snRNPs). Alternative splicing allows one gene to produce multiple protein isoforms.
• Translation in Detail: Initiation involves the small ribosomal subunit binding the mRNA and scanning for the first AUG start codon. Elongation is a three-step cycle: 1) aminoacyl-tRNA binding to the A site, 2) peptide bond formation (catalyzed by rRNA - a ribozyme), 3) ribosome translocation. Termination occurs when a stop codon is recognized by a release factor.

2.1.1 Eukaryotic RNA Polymerases (I, II, III)

• RNA Polymerase I: Located in the nucleolus, transcribes most rRNA genes.
• RNA Polymerase II: Located in the nucleoplasm, transcribes all mRNA genes (protein-coding) and some non-coding RNAs (e.g., snRNAs, miRNAs).
• RNA Polymerase III: Located in the nucleoplasm, transcribes tRNA genes, the 5S rRNA gene, and other small RNAs.

2.1.2 The Ribosome: A-P-E Sites

The ribosome (80S in eukaryotes, 70S in prokaryotes) has three key sites for tRNA:

• A (Aminoacyl) site: The "entry" point, where a new, charged aminoacyl-tRNA binds.
• P (Peptidyl) site: The "processing" point, where the tRNA holding the growing polypeptide chain is located.
• E (Exit) site: The "exit" point, where the uncharged tRNA is ejected from the ribosome.

2.2 Gene Regulation and Post-Translational Modifications

2.2.1 Chromatin Remodeling

Gene expression in eukaryotes is fundamentally controlled by chromatin structure.

• Euchromatin: A loosely packed, "open" state of chromatin. Histones are typically acetylated, which neutralizes their positive charge and reduces their affinity for DNA. This state is permissive for transcription.
• Heterochromatin: A tightly condensed state. Histones are typically deacetylated and methylated (at specific residues like H3K9). This state is transcriptionally silent.

2.2.2 Expanding the World of Non-coding RNA

While miRNAs are well-known for post-transcriptional silencing, other non-coding RNAs are also critical:

• Long non-coding RNAs (lncRNAs): Over 200 nucleotides, they act as scaffolds, decoys, or guides for chromatin-modifying complexes, influencing gene expression on a large scale.
• Small interfering RNAs (siRNAs): Often derived from external sources (like viruses) or experimental manipulation (RNAi), they use the same machinery as miRNAs to perfectly bind and cleave a target mRNA, leading to potent gene silencing.

Part 3: Cellular Compartments, Cytoskeleton, and Cell-Cell Interactions

This section explores the specialized compartments, structural framework, and intercellular connections that define a eukaryotic cell and its integration into tissues.

3.1 The Endomembrane System and Intracellular Trafficking

• The Secretory Pathway: RER → Golgi → Vesicles → Final Destination. Vesicular transport between compartments is mediated by coat proteins like COP I (retrograde, Golgi to ER) and COP II (anterograde, ER to Golgi).
• Endocytosis and the Endosome-Lysosome Pathway: Material is internalized via endocytosis. Receptor-mediated endocytosis is highly specific, involving receptors that cluster in clathrin-coated pits (e.g., uptake of LDL). Internalized vesicles fuse with early endosomes, which act as sorting stations. From here, receptors can be recycled to the plasma membrane, while cargo is trafficked to late endosomes and finally to lysosomes for degradation.

3.1.1 The Smooth ER and Detoxification

While the Rough ER is studded with ribosomes for protein synthesis, the Smooth ER (SER) lacks ribosomes and has distinct, crucial functions. It is the primary site of lipid synthesis (including steroids and phospholipids) and calcium storage. A key function, especially in liver cells, is detoxification, carried out by the cytochrome P450 family of enzymes, which make hydrophobic toxins more water-soluble for excretion.

3.1.2 Protein Sorting and Signal Peptides

How does a cell "know" where a protein belongs? The protein itself carries a "zip code" in its amino acid sequence called a signal peptide (or signal sequence).

• ER Signal Sequence: An N-terminal hydrophobic sequence directs the ribosome to the RER (co-translational import).
• Nuclear Localization Signal (NLS): A short, positively charged sequence allows proteins (like histones) to be actively imported into the nucleus.
• Mitochondrial Targeting Sequence: An N-terminal amphipathic helix directs proteins to the mitochondria.

3.2 Specialized Organelles and Signal Transduction

• Mitochondria: Beyond ATP: They possess their own circular DNA and are central to calcium homeostasis, synthesis of heme groups, and the intrinsic pathway of apoptosis via release of cytochrome c.
• Signal Transduction: Cells respond to external signals via receptors. G-protein coupled receptors (GPCRs) activate intracellular signaling cascades through second messengers like cAMP and Ca²⁺. Receptor tyrosine kinases (RTKs) dimerize upon ligand binding and autophosphorylate, creating docking sites for downstream signaling proteins, often activating the MAP kinase pathway which regulates gene expression and cell proliferation.

3.2.1 Peroxisomes: The Oxidation Specialist

Peroxisomes are small, membrane-enclosed organelles that contain oxidative enzymes. Their key functions include:

1. Breakdown of very-long-chain fatty acids (VLCFAs) via beta-oxidation.
2. Detoxification of harmful substances (e.g., ethanol).
3. Neutralizing Reactive Oxygen Species (ROS): They produce hydrogen peroxide ($H_2O_2$) as a byproduct of oxidation but contain the enzyme catalase to safely break it down into water and oxygen.

3.2.2 Intracellular (Steroid) Receptors

Not all signals are received at the cell surface. Small, hydrophobic molecules like steroid hormones (e.g., cortisol, estrogen) can diffuse directly across the plasma membrane. They bind to intracellular receptors (or nuclear receptors) located in the cytosol or nucleus. Upon binding, the receptor-hormone complex undergoes a conformational change, translocates to the nucleus, and binds directly to specific DNA sequences (Hormone Response Elements) to act as a transcription factor, directly altering gene expression.

3.3 The Cytoskeleton: Form and Function

The cytoskeleton provides the cell with structural integrity, facilitates movement, and organizes intracellular transport. It is composed of three main types of filaments.

3.4 Cell Junctions and Tissue Architecture

Part 4: Key Molecular Biology Techniques

A crucial part of modern medicine is the ability to analyze and manipulate the molecules of life. This section reviews the core laboratory techniques essential for diagnostics and research.

4.1 Amplifying DNA: Polymerase Chain Reaction (PCR)

PCR is a revolutionary technique used to exponentially amplify a specific segment of DNA. It relies on a three-step cycle repeated 25-35 times:

1. Denaturation (95°C): The high temperature separates the two strands of the target DNA.
2. Annealing (55-65°C): The temperature is lowered to allow short DNA primers, complementary to the target sequence, to bind (anneal).
3. Extension (72°C): A heat-stable DNA polymerase (like Taq polymerase) synthesizes new DNA strands, starting from the primers.

4.2 Separating Molecules: Gel Electrophoresis

This technique separates molecules based on their size (and sometimes charge) by moving them through a gel matrix using an electric field.

• Agarose Gel Electrophoresis: Used for separating large DNA fragments. Since DNA is negatively charged (due to phosphate groups), it moves towards the positive anode. Smaller fragments migrate faster and further.
• SDS-PAGE (Polyacrylamide Gel Electrophoresis): Used for separating proteins. The detergent SDS denatures proteins and gives them a uniform negative charge. Therefore, proteins are separated almost exclusively by their size (mass).

4.3 Manipulating DNA: Recombinant DNA Technology

This involves combining DNA from different sources. The key tools are:

• Restriction Enzymes (Endonucleases): Bacterial enzymes that recognize and cut specific, often palindromic, DNA sequences. Many create "sticky ends" (overhangs).
• Plasmids: Small, circular DNA molecules found in bacteria that are used as "vectors" to carry a gene of interest.
• DNA Ligase: The same enzyme from replication is used to "paste" the gene of interest (cut with the same restriction enzyme) into the opened plasmid vector.

4.4 Detecting Molecules: Blotting Techniques

Blotting involves transferring molecules from a gel to a solid membrane, followed by detection using a specific probe.

4.5 Editing the Genome: CRISPR-Cas9

The CRISPR-Cas9 system is a powerful genome-editing tool derived from a bacterial immune system. It consists of two components:

• Cas9: An endonuclease (an enzyme that cuts DNA).
• guide RNA (gRNA): A short, engineered RNA molecule that "guides" the Cas9 enzyme to a specific target sequence in the genome.

Interactive Practice Quiz

Rigorously test your understanding of these advanced concepts. Choose the best answer for each question and then submit to see your results.