DNA Replication: Process and Mechanism

DNA Replication

• A cell duplicates its DNA through DNA replication.
• This ensures genetic information is passed to daughter cells.
• DNA replication is semi-conservative.
• Each new DNA molecule has one original (parental) and one newly synthesized strand.
• Base pairing is the hydrogen bonding between purines and pyrimidines.
• Adenine (A) pairs with thymine (T), and guanine (G) pairs with cytosine (C).
• A DNA template strand guides the synthesis of a complementary strand during replication.
• DNA polymerase adds nucleotides to the growing DNA strand, working 5' to 3'.
• Replication starts at a specific DNA sequence called the replication origin.
• This allows the DNA to unwind and the replication machinery to assemble.
• The replication fork is a Y-shaped structure where the double helix unwinds, and new strands are synthesized.
• The leading strand is synthesized continuously in the 5' to 3' direction toward the replication fork.
• The lagging strand is synthesized discontinuously in short Okazaki fragments.
• Synthesis occurs in the 5' to 3' direction away from the replication fork.
• A primer is a short RNA sequence which provides a starting point for DNA polymerase, and is synthesized by primase.
• Primase synthesizes RNA primers that are needed to start DNA replication.
• Okazaki fragments are short DNA fragments synthesized on the lagging strand during DNA replication.
• They are later joined by DNA ligase to form a continuous strand.
• DNA ligase joins Okazaki fragments on the lagging strand.
• This is achieved by forming phosphodiester bonds, creating a continuous DNA strand.
• The 5' end has a phosphate group, and the 3' end has a hydroxyl group.
• DNA synthesis occurs in the 5' to 3' direction.
• DNA polymerase can remove incorrectly paired nucleotides during DNA replication.
• This ensures high fidelity through DNA Polymerase Proofreading.
• DNA Helicase unwinds the DNA double helix at the replication fork.
• This separates the two strands.
• Single-Strand DNA Binding Proteins (SSBs) proteins bind to separated DNA strands.
• This prevents them from reannealing or forming secondary structures.
• A sliding clamp holds DNA polymerase in place during replication.
• This ensures efficient synthesis.
• A clamp loader loads the sliding clamp onto DNA, facilitating the attachment of DNA polymerase during replication.
• Topoisomerase relieves tension caused by DNA unwinding ahead of the replication fork.
• It achieves this by cutting and rejoining the DNA strands.
• Telomeres are repetitive nucleotide sequences at the ends of chromosomes.
• They protect them from deterioration during replication.
• Telomerase extends telomeres with repetitive nucleotide sequences.
• This counteracts shortening during replication.
• Depurination is the loss of a purine base (adenine or guanine) from the DNA molecule.
• This can lead to potential mutations if not repaired.
• Deamination is the removal of an amino group from a nucleotide base.
• An example is the conversion of cytosine to uracil in DNA.
• Mismatch Repair is a DNA repair mechanism that corrects mismatched nucleotides incorporated during DNA replication.
• Double-Strand Break RepairCellular mechanisms, like homologous recombination and non-homologous end joining, repair breaks affecting both strands of the DNA double helix.

Gene Expression, Transcription, and Translation

• Gene expression is the process by which information from a gene is used to synthesize functional gene products.
• Transcription is the synthesis of RNA from a DNA template, where the DNA sequence is copied into a complementary RNA sequence.
• Translation is the process by which ribosomes synthesize proteins using the mRNA transcript produced during transcription.
• DNA contains deoxyribose sugar and uses thymine (T).
• RNA contains ribose sugar and uses uracil (U).
• RNA transcript is complementary to the DNA template strand, which is made through transcription.
• RNA folding is the process by which an RNA molecule folds into its three-dimensional structure, often forming secondary structures like hairpins.
• Complementary strands are strands of DNA or RNA that can form base pairs with each other.
• In DNA, adenine (A) pairs with thymine (T) and guanine (G) pairs with cytosine (C).
• In RNA, adenine (A) pairs with uracil (U).
• RNA Polymerase synthesizes RNA from a strand of DNA.
• There are several types of RNA: messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), and microRNA (miRNA) (non-coding RNA).
• Messenger RNA (mRNA) carries genetic information from DNA to the ribosome
• At the ribosome, it specifies the amino acid sequence of protein products.
• Ribosomal RNA (rRNA) forms the core of the ribosome's structure.
• It catalyzes protein synthesis.
• MicroRNA (miRNA) regulates gene expression.
• Transfer RNAs (tRNA) serve as adaptors between mRNA and amino acids during protein synthesis.
• Noncoding RNA is used in RNA splicing, gene regulation, telomere maintenance, and other processes.
• A promoter is a DNA sequence that signals the start site for transcription and the binding site for RNA polymerase.
• In eukaryotes, main types of RNA Polymerases are: RNA Polymerase I (synthesizes rRNA), RNA Polymerase II (synthesizes mRNA and some snRNA), and RNA Polymerase III (synthesizes tRNA and some other small RNAs).
• A TATA box is a DNA sequence found in many eukaryotic promoters.
• It indicates where a genetic sequence can be read and decoded.
• TFIID is a transcription factor that recognizes and binds to the TATA box.
• This plays a critical role in initiating transcription.
• Transcription factors are proteins that help turn specific genes on or off by binding to nearby DNA.
• Capping factors add the 5' cap to the nascent RNA transcript
• This is essential for mRNA stability and initiation of translation.
• Proteins remove introns from pre-mRNA to produce mature mRNA.
• Polyadenylation factors add a poly(A) tail to the 3' end of the mRNA transcript.
• This enhances stability and export from the nucleus.
• Eukaryotic mRNA Processing are the modifications that a pre-mRNA undergoes to become mature mRNA.
• Examples include capping, splicing, and polyadenylation.
• Introns are non-coding sequences in a gene that are removed during RNA splicing.
• Exons are coding sequences in a gene that remain in the mRNA after splicing and are expressed as protein.
• RNA splicing removes introns and joining exons in a pre-mRNA to produce mature mRNA.
• Alternative splicing generates different forms of mature mRNAs (and thus different proteins)
• It achieves this from the same gene by splicing the pre-mRNA in different ways.
• Mature RNA export transports fully processed mRNA from the nucleus to the cytoplasm for translation.
• Genetic code is the set of rules by which information encoded in mRNA sequences is translated into proteins by living cells.
• A codon is a sequence of three nucleotides in mRNA that specifies a particular amino acid or termination signal.
• A reading frame is how nucleotides in mRNA are grouped into codons.
• There are three possible reading frames for any sequence.
• Amino acid codons are specific sequences of three nucleotides (codons) in mRNA that correspond to specific amino acids.
• Membrane-Bound Organelles are specialized structures within eukaryotic cells.
• Examples include the nucleus, endoplasmic reticulum, mitochondria, and chloroplasts, enclosed by lipid bilayers that compartmentalize cellular functions.

Cellular Structures and Processes

• The nucleus is believed to have originated from the invagination of the plasma membrane.
• This forms a protective compartment around the genetic material.
• The endoplasmic reticulum (ER) is thought to have evolved from the extension of the nuclear envelope.
• This creates a network of membranes involved in protein and lipid synthesis.
• Mitochondria are believed to have originated from an endosymbiotic event.
• An ancestral eukaryotic cell engulfed an aerobic prokaryote, leading to a symbiotic relationship.
• Chloroplasts are thought to have arisen from an endosymbiotic event.
• A eukaryotic cell engulfed a photosynthetic cyanobacterium, leading to a symbiotic relationship.
• Types of protein transport are: gated transport (through nuclear pores), transmembrane transport (across organelle membranes), and vesicular transport (via membrane-bound vesicles).
• A signal sequence is a short amino acid sequence that directs a protein to its correct location in the cell. e.g nucleus, mitochondria, or ER
• Nuclear import is the process by which proteins with a nuclear localization signal are transported into the nucleus through nuclear pore complexes.
• Mitochondrial import translocates of proteins into mitochondria.
• It requires specific signal sequences and translocator complexes in the mitochondrial membranes.
• Synthesis of Soluble Proteins into the ER Lumen is the co-translational process in which ribosomes synthesize proteins directly into the ER lumen, guided by an ER signal sequence.
• Vesicular transport moves proteins and lipids between organelles.
• This is achieved in membrane-bound vesicles that bud from one compartment and fuse with another.
• The secretory pathway processes proteins synthesized in the ER in the Golgi apparatus.
• These are transported to the plasma membrane or extracellular space.
• Exocytosis is the process by which vesicles fuse with the plasma membrane to release their contents outside the cell.
• Endocytosis uptakes of external substances by the inward folding of the plasma membrane.
• This forms vesicles that transport the substances into the cell.
• An endosome is a membrane-bound compartment inside eukaryotic cells.
• It sorts and directs vesicular traffic, especially during endocytosis.
• A lysosome is an organelle containing digestive enzymes that break down waste materials, cellular debris, and foreign pathogens.
• Clathrin is a protein that forms a triskelion shape and assembles into a polyhedral lattice.
• This is necessary for the formation of coated vesicles during endocytosis.
• v-SNAREs are vesicle-associated membrane proteins that mediate the fusion of transport vesicles with target membranes by pairing with t-SNAREs.
• t-SNAREs are target membrane proteins that pair with v-SNAREs to facilitate the docking and fusion of vesicles with their target membranes.
• A tethering protein initially captures transport vesicles by binding to Rab proteins on the vesicle surface.
• This facilitating vesicle docking.
• Docking is when a transport vesicle is positioned and held at the target membrane prior to fusion.
• This is mediated by interactions between SNAREs and tethering proteins.
• The Golgi apparatus organizes a stack of membrane-bound cisternae.
• It modifies, sorts, and packages proteins and lipids received from the ER for delivery to various destinations.
• Constitutive secretion is a continuous, unregulated process.
• Proteins and lipids are secreted from the cell as soon as they are synthesized and processed.
• Regulated secretion stores proteins in secretory vesicles.
• It releases them from the cell in response to specific signals or stimuli.
• Selective permeability is the property of cellular membranes that allows certain molecules to pass through while restricting others.
• This allows the cell to maintain a distinct internal environment.
• A concentration gradient is a difference in the concentration of a substance across a space or membrane.
• It drives passive transport processes.
• Membrane potential is the voltage difference across a cell's plasma membrane.
• This results from the distribution of ions, influencing the movement of charged substances.
• Channels are protein structures that form pores in the membrane, allowing specific ions or molecules to pass through by diffusion.
• Transporters are membrane proteins that bind to specific molecules.
• They undergo conformational changes to shuttle them across the membrane
• Simple diffusion passively moves molecules from an area of higher concentration to an area of lower concentration without the assistance of membrane proteins.
• Passive transport moves substances across a membrane down their concentration or electrochemical gradient without using cellular energy.
• Active transport moves molecules across a membrane.
• Transport occurs against their concentration or electrochemical gradient, requiring energy input, typically from ATP.
• Pumps transport substances against their concentration gradient.
• They use energy to move substances across a membrane
• Electrochemical gradient is the combined effect of an ion's concentration gradient and the membrane potential.
• This influences its movement across the membrane.
• Osmosis is a passive diffusion of water molecules across selectively permeable membrane.
• Diffusion occurs from a region of lower solute concentration to one of higher solute concentration.
• Aquaporins are specialized channel proteins that facilitate the rapid transport of water molecules across the cell membrane.
• "With the gradient" describes movement of substances from areas of higher concentration to areas of lower concentration.
• Movement is following the natural direction of the concentration gradient.
• "Against the gradient" describes movement of substances from areas of lower concentration to areas of higher concentration.
• Movement is opposing the natural direction of the concentration gradient, requiring energy input.
• Gradient-driven pumps are transport proteins that couple the movement of one molecule down its gradient to drive the movement of another molecule against its gradient.
• ATP-driven pumps are transporters that use the energy from ATP hydrolysis.
• The energy is used to move molecules against their concentration gradients.
• Light-driven pumps are transport proteins that use energy from light.
• The energy is used to move substances across membranes against their concentration gradients.
• The Na+/K+ pump is an ATP-driven pump for active transport.
• It actively transports three sodium ions (Na⁺) out and two potassium ions (K⁺) into the cell, for maintenance of concentration gradients.
• Ion channel selectivity allows ion channels to permit the passage of specific ions while excluding others.
• Exclusion is based on factors like size and charge.
• Channel gating controls how ion channels open or close in response to specific stimuli.
• This then regulates ion flow across the membrane.
• Three subcategories of gating modulate ion channels.
• Gating occurs via voltage changes (voltage-gated), ligand binding (ligand-gated), or mechanical forces (mechanically-gated), each related to different types of stimuli.