DNA Replication, Gene Expression, and Cell Division: Study Guide for BIO 043

DNA Replication: Mechanisms and Enzymes

5′ to 3′ Synthesis and Strand Polarity

DNA replication proceeds in the 5′ to 3′ direction, requiring a free 3′-OH group for nucleotide addition. The antiparallel nature of DNA results in continuous synthesis on the leading strand and discontinuous synthesis on the lagging strand.

• Leading Strand: Synthesized continuously toward the replication fork.

• Lagging Strand: Synthesized discontinuously away from the fork, forming Okazaki fragments.

• Free 3′-OH Requirement: DNA polymerases can only add nucleotides to an existing 3′-OH group.

Key Enzymes in DNA Replication

• DNA Polymerase III: Main enzyme for synthesizing new DNA strands in prokaryotes; high processivity and proofreading activity.

• DNA Polymerase I: Removes RNA primers and fills in gaps with DNA nucleotides.

• DNA Helicase: Unwinds the double helix at the replication fork.

• Primase: Synthesizes short RNA primers to provide a starting point for DNA synthesis.

• Topoisomerase: Relieves supercoiling tension ahead of the replication fork.

• Single-Stranded Binding Proteins (SSBs): Stabilize unwound DNA strands, preventing re-annealing.

Okazaki Fragments and DNA Ligase

• Okazaki Fragments: Short DNA segments synthesized on the lagging strand.

• DNA Ligase: Joins Okazaki fragments by forming phosphodiester bonds between adjacent DNA segments.

Telomerase and Linear Chromosome Replication

• Telomerase: An enzyme that extends telomeres (repetitive DNA at chromosome ends) to prevent loss of genetic information during replication.

• Importance: Maintains chromosome integrity, especially in germ cells and stem cells.

Transcription and mRNA Processing

Transcription Directionality and Template Reading

• Template Strand: DNA is read 3′ → 5′ to synthesize mRNA 5′ → 3′.

• mRNA Transcript: Complementary to the DNA template strand.

Untranslated Regions (UTRs)

• 5′ and 3′ UTRs: Non-coding regions flanking the coding sequence; regulate mRNA stability, localization, and translation efficiency.

Eukaryotic mRNA Maturation

• 5′ Methylguanosine Capping: Protects mRNA from degradation and aids in ribosome binding.

• Splicing: Removal of introns and joining of exons by the spliceosome.

• Poly-A Tail: Addition of a string of adenine nucleotides at the 3′ end, enhancing mRNA stability and export.

The Genetic Code and the Wobble Hypothesis

Properties of the Genetic Code

• Redundancy/Degeneracy: Multiple codons can specify the same amino acid.

• Start Codon: AUG (methionine) signals translation initiation.

• Stop Codons: UAA, UAG, UGA signal translation termination.

Wobble Hypothesis and Inosine

• Wobble Hypothesis: Flexibility in base pairing at the third codon position allows one tRNA to recognize multiple codons.

• Inosine (I): A modified base in tRNA anticodons that can pair with U, C, or A in mRNA.

Translation: Ribosome Structure and Energetics

Ribosomal Sites and Sequences

• A Site (Aminoacyl): Entry point for charged tRNAs.

• P Site (Peptidyl): Holds the tRNA with the growing polypeptide chain.

• E Site (Exit): Where uncharged tRNAs exit the ribosome.

• Peptidyl Transferase: Catalyzes peptide bond formation.

• Kozak Sequence (Eukaryotes): Consensus sequence for translation initiation.

• Shine-Dalgarno Sequence (Prokaryotes): Ribosome binding site upstream of the start codon.

Translation Energetics

• ATP and GTP Expenditure: High-energy phosphate bonds are used for amino acid charging (ATP), initiation, elongation, and termination (GTP).

Mutations and mRNA Surveillance

Types of Mutations

• Missense Mutation: Alters a single amino acid in the protein.

• Nonsense Mutation: Introduces a premature stop codon, truncating the protein.

• Frameshift Mutation: Insertions or deletions that shift the reading frame, altering downstream amino acids.

Nonsense-Mediated Decay (NMD) and Exon Junction Complexes (EJCs)

• NMD: Cellular mechanism that degrades mRNAs containing premature stop codons.

• Exon Junction Complexes: Protein complexes deposited at exon-exon junctions during splicing; their position relative to stop codons helps trigger NMD.

DNA Double-Strand Break (DSB) Repair Mechanisms

Repair Pathways

Pathway	Template Requirement	Fidelity	Mechanism
Non-Homologous End Joining (NHEJ)	No	Low (error-prone)	Direct ligation of broken ends
Synthesis-Dependent Strand Annealing (SDSA)	Yes	High	Uses homologous sequence for repair, no crossover
Homologous Recombination (HR)	Yes	High	Uses homologous chromosome or sister chromatid, can result in crossover

Cell Cycle Phases and Chromosome Structure

Cell Cycle Phases

• G1 Phase: Cell growth and preparation for DNA synthesis.

• S Phase: DNA replication; chromosomes duplicate.

• G2 Phase: Preparation for mitosis; error checking.

• M Phase: Mitosis and cytokinesis; cell division.

Chromatin, Chromosomes, and Chromatids

• Chromatin: DNA-protein complex; less condensed form found during interphase.

• Chromosome: Highly condensed DNA visible during mitosis/meiosis.

• Sister Chromatids: Identical copies of a chromosome, joined at the centromere after DNA replication.

• Homologous Chromosomes: Chromosome pairs (one from each parent) with the same genes but possibly different alleles.

Mitosis and Cytokinesis

Chromosome Behavior and Cell Division Structures

• Prophase to Telophase: Chromosomes condense, align, separate, and decondense.

• Karyokinesis: Division of the nucleus.

• Cytokinesis: Division of the cytoplasm; cleavage furrow in animals, cell plate in plants.

Spindle Assembly Checkpoint (SAC) and Chromosome Segregation

Kinetochore Attachment and Molecular Cascade

• Biorientation/Amphitelic Attachment: Each sister chromatid attaches to microtubules from opposite spindle poles, generating tension.

• Molecular Cascade: Mad/Bub proteins inhibit APC/C until proper attachment; APC/C activation leads to Securin degradation, releasing Separase to cleave Cohesin and allow chromatid separation.

Meiosis: Stages and Genetic Diversity

Meiosis I vs. Meiosis II

• Meiosis I: Homologous chromosomes separate; reductional division.

• Meiosis II: Sister chromatids separate; equational division.

Prophase I Sub-stages (in order)

1. Leptotene

2. Zygotene

3. Pachytene

4. Diplotene

5. Diakinesis

Chromosome Separation and Cohesin Protection

• Anaphase I: Homologous chromosomes separate.

• Anaphase II: Sister chromatids separate.

• Shugoshin (Sgo1): Protects centromeric cohesin during Meiosis I, ensuring proper chromatid cohesion until Meiosis II.

Sources of Genetic Diversity

• Crossing Over: Exchange of genetic material between homologous chromosomes at chiasmata (tetrads).

• Independent Assortment: Random orientation of homologous pairs during Metaphase I.

• Random Fertilization: Any sperm can fertilize any egg, increasing genetic variation.

Chromosomal Aberrations: Nondisjunction and Aneuploidy

• Nondisjunction: Failure of homologous chromosomes or sister chromatids to separate properly during meiosis.

• Aneuploidy: Resulting gametes have abnormal chromosome numbers (e.g., trisomy, monosomy).

Mendelian Genetics: Test Crosses

Principles and Applications

• Test Cross: Crossing an individual with a dominant phenotype (unknown genotype) to a homozygous recessive individual to determine genotype.

• Interpretation: Offspring phenotypes reveal whether the dominant parent is homozygous or heterozygous.

Example: If all offspring display the dominant trait, the parent is likely homozygous dominant; if a 1:1 ratio of dominant to recessive appears, the parent is heterozygous.