DNA Replication Quiz

High-Yield Principles in Biochemistry

• Biochemistry is the study of carbon compounds.
• Fundamental building blocks of life are made in collapsing stars.
• The power to control genetics is awesome and terrifying.
• Studying metabolic pathways, enzyme deficiencies and regulatory steps are important for high yield.
• This study material includes topics in molecular biology, genetics, cell biology, metabolic principles (vitamins, cofactors, minerals, single-enzyme deficiency diseases).
• Don't spend time learning details about organic, physical, and chemical mechanisms or structures.
• Understanding biochemical techniques such as ELISA, immunoelectrophoresis, Southern blotting, and PCR is important.
• Integrating biochemistry with pharmacology and genetics is valuable.

Chromatin Structure

• DNA exists in chromatin form to fit into the nucleus.
• DNA loops around a histone octamer to form a nucleosome.
• Hl binds to nucleosomes and linker DNA, which stabilizes the chromatin fiber.
• DNA has a negative charge, whereas histones are positive.
• In mitosis, DNA condenses into chromosomes.
• DNA and histone synthesis occurs during S phase.
• Mitochondria have circular DNA without histones.

Nucleotides

• Nucleoside = base + sugar (ribose/deoxyribose)
• Nucleotide = base + sugar (ribose/deoxyribose) + phosphate, linked by 3'-5' phosphodiester bonds.
• Purines (adenine, guanine) have two rings.
• Pyrimidines (cytosine, uracil, thymine) have one ring.
• Uracil is in RNA, and thymine is in DNA.
• Methylation of uracil forms thymine.
• The 5' end of a nucleotide has a triphosphate group that is the energy source.

De Novo Pyrimidine and Purine Synthesis

• Various drugs (e.g., immunosuppressants, antineoplastics, antibiotics) work by interfering with nucleotide synthesis.
• Examples of drugs that interfere are 6-MP, MTX, Azathioprine, Mycophenolate, and Ribavirin.
• Leflunomide inhibits dihydroorotate dehydrogenase.

Purine Salvage Deficiencies

• Adenosine deaminase (ADA) deficiency affects the degradation of adenosine and deoxyadenosine.
• This leads to increased dATP, which inhibits ribonucleotide reductase activity and DNA precursors in cells. Decreases lymphocytes.
• Defective purine salvage (absent HGPRT) results in increased uric acid production
• Lesch-Nyhan syndrome is an X-linked recessive trait caused by deficient HGPRT activity.
• Characterized by intellectual disability, self-mutilation, aggression, hyperuricemia (↑ uric acid), gout and dystonia.
• Treatment involves allopurinol and febuxostat.

DNA Replication

• DNA replication occurs in a 5' to 3' direction.
• It is semiconservative (each new DNA molecule contains one original strand and one new strand).
• Starts at origins of replication.
• Helicase unwinds DNA at the replication fork.
• Single-stranded binding proteins (SSBs) prevent strands from reannealing.
• Topoisomerases relieve DNA supercoiling.
• Primase synthesizes RNA primers.
• DNA polymerase III elongates the leading and lagging strands.
• DNA polymerase I removes RNA primers and replaces them with DNA.
• DNA ligase joins Okazaki fragments.
• Telomerase adds repetitive sequences to telomeres to prevent loss of genetic material during replication.

DNA Repair

• Double-strand breaks repaired by nonhomologous end joining (NHEJ) or homologous recombination.
• Nucleotide excision repair: removes bulky helix-distorting lesions (e.g., pyrimidine dimers).
• Base excision repair: removes altered bases and apurinic/apyrimidinic (AP) sites.
• Mismatch repair fixes mismatched nucleotides in the new strand.

Mutations in DNA

• Single nucleotide substitutions include transitions (purine to purine or pyrimidine to pyrimidine) and transversions (purine to pyrimidine or vice versa).
• Frameshift mutations result from insertions or deletions of nucleotides that are not multiples of 3.
• Splice site mutations lead to retained introns in mRNA, affecting protein function.

The Lac Operon

• The lac operon (in E. coli) regulates lactose metabolism.
• Low glucose → ↑ cAMP → activation of CAP protein → increased transcription.
• High lactose → repressor protein detaches from operator (gene) site → increased transcription

Functional Organization of a Eukaryotic Gene

• A gene contains coding regions (exons) and non-coding regions (introns).
• Promoters are regions where RNA polymerase binds and initiates transcription.
• Enhancers and silencers affect gene expression.

RNA Processing (Eukaryotes)

• Initial transcript (hnRNA) is modified to become mRNA.
• Capping of the 5' end, polyadenylation of the 3' end, and splicing of introns.
• Mature mRNA is transported to the cytoplasm to be translated.
• Quality control of mRNA occurs in processing bodies (P-bodies).

Introns vs exons

• Exons contain the code for proteins or functional RNA.
• Introns are non-coding sequences, important for gene regulation.
• Alternative splicing combines different exons to produce multiple proteins from a single gene.

tRNA Structure and Charging

• tRNA is cloverleaf-shaped (75-90 nucleotides) with a CCA 3' end
• Aminoacyl-tRNA synthetases are enzymes responsible for attaching the correct amino acid to a tRNA
• Anticodon loop recognizes a codon.
• There are unique enzymes (1 per amino acid) to pair a specific amino acid with its tRNA.

Protein Synthesis

• Protein synthesis occurs in the cytoplasm/ribosomes.
• Initiation factors help attach the 40S ribosomal subunit to the mRNA.
• Elongation factors add amino acids to the growing polypeptide chain.
• Termination factors stop polypeptide synthesis when a stop codon is reached.
• Post-translational modifications alter the protein structure (eg. trimming, covalent alteration, or chaperone proteins.)

Cell Cycle Phases

• Checkpoints regulate the progression through the cell cycle (G1, S, G2, M)
• Cyclins and cyclin-dependent kinases (CDKs) regulate the cell cycle progression.
• Tumor suppressors (eg. p53, Rb) play a role in regulating cell cycle progression.

Rough and Smooth Endoplasmic Reticulum

• Rough ER (RER) synthesizes secretory proteins and adds N-linked oligosaccharides to lysosomal proteins.
• RER location is inside the cytoplasm of cells; ribosomes embedded on the outer membrane.
• Smooth ER (SER) synthesizes steroid hormones and helps detoxify drugs and poisons.
• SER location is inside the cytoplasm of cells; free ribosomes are not attached to the membrane.

Cell Trafficking

• Golgi apparatus sorts and modifies proteins and lipids from the ER.
• Endosomes sort material from the exterior and Golgi to lysosomes or back to the membrane/Golgi
• I-cell disease is a lysosomal storage disorder caused by a defect in N-acetylglucosaminyl-1-phosphotransferase.

Cytoskeletal Elements

• Microfilaments (made of actin): cell contraction.
• Intermediate filaments: maintain cell structure (eg. vimentin.)
• Microtubules: essential for cell division, transport, and cilia/flagella.
• Molecular motors (dyneins and kinesins): transport to different directions/ends on a microtubule track

Peroxisome

• Membrane-enclosed organelle involved in ß-oxidation of very long-chain fatty acids, a-oxidation of branched-chain fatty acids.
• Catabolism of amino acids and ethanol.
• Synthesis of bile acids and plasmalogens (important membrane lipids, especially in white matter).

Proteasome

• Barrel-shaped protein complex involved in degrading polyubiquitin-tagged proteins.
• Many cellular processes depend on the proteasome such as the immune response, neurodegenerative disorders.

DNA Repair

• Non-homologous end joining (NHEJ)- two damaged ends are joined.
• Homologous recombination- a damaged strand is repaired using a complementary strand.
• Nucleotide excision repair- removes pyrimidine dimers formed by UV damage.
• Base excision repair- removes altered bases or apurinic/apyrimidinic sites.
• Mismatch repair- corrects errors missed during DNA replication

DNA Repair mechanisms

• Cells use various mechanisms to repair damaged DNA, ensuring genetic integrity.
• NHEJ repairs double-strand breaks.
• Homologous recombination ensures accuracy in DNA strand repair.