Chapter 17: DNA Replication, Repair, and Recombination

Stages of the Eukaryotic Cell Cycle

• G1 Phase: Cell grows and prepares for DNA replication.

• S Phase: DNA synthesis (replication) occurs.

• G2 Phase: Cell prepares for mitosis; checks for DNA errors.

• M Phase: Mitosis and cytokinesis; cell divides into two daughter cells.

DNA Replication: Models and Mechanisms

• Watson-Crick Model: DNA is a double helix with complementary base pairing (A-T, G-C).

• Semiconservative Replication: Each daughter DNA molecule contains one parental and one new strand. Demonstrated by the Meselson-Stahl experiment using isotopic labeling.

Steps and Components of DNA Replication

• Pre-replication Complex (pre-RC): Assembles at origins of replication during G1 phase.

• Initiation: Origin recognition, DNA unwinding, and recruitment of replication machinery.

• DNA Synthesis: DNA polymerases synthesize new DNA strands in the 5' to 3' direction.

• Replisome: Multiprotein complex that coordinates DNA replication.

• Directionality: DNA synthesis always proceeds 5' to 3'.

Key Enzymes and Proteins in Replication

• DNA Polymerases: Catalyze DNA synthesis; require a primer and template.

• Helicases: Unwind the DNA double helix.

• Topoisomerases: Relieve supercoiling ahead of the replication fork.

• Single-Stranded Binding Proteins (SSB/RPA): Stabilize unwound DNA.

• Primase: Synthesizes short RNA primers for DNA polymerase to extend.

• MCM Complex: Eukaryotic helicase essential for DNA unwinding.

• PCNA: Sliding clamp that increases DNA polymerase processivity.

• DNA Polymerase III: Main bacterial replicative polymerase.

• DNA Polymerase α, δ, ε: Eukaryotic polymerases with distinct roles in replication.

Replication Fork and Bubble

• Replication Fork: Y-shaped region where DNA is actively unwound and replicated.

• Replication Bubble: Region of locally unwound DNA where replication occurs.

Leading and Lagging Strands

• Leading Strand: Synthesized continuously in the direction of fork movement.

• Lagging Strand: Synthesized discontinuously as Okazaki fragments, later joined by DNA ligase.

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

Proofreading and Fidelity

• DNA Polymerase Proofreading: 3'→5' exonuclease activity removes misincorporated nucleotides, increasing fidelity.

End Replication Problem and Telomeres

• End Replication Problem: Inability to fully replicate linear chromosome ends.

• Telomeres: Repetitive DNA sequences at chromosome ends.

• Telomerase: Enzyme that extends telomeres using an RNA template.

Mutations: Types and Causes

• Mismatch: Incorrect base pairing during replication.

• Trinucleotide Repeats: Expansion of short, repeated sequences (e.g., Huntington's disease).

• Base Modifications: Chemical changes to bases (e.g., methylation, oxidation).

• Depurination: Loss of a purine base (A or G).

• Deamination: Removal of an amino group from a base (e.g., cytosine to uracil).

DNA Repair Pathways

• Base Excision Repair (BER): Removes and replaces damaged bases.

• Mismatch Repair (MMR): Corrects replication errors not fixed by proofreading.

• Nucleotide Excision Repair (NER): Removes bulky DNA lesions (e.g., thymine dimers).

Translesion DNA Synthesis

• Process: Specialized polymerases bypass DNA lesions but are error-prone.

Double-Strand Break Repair

• Homologous Recombination (HR): Uses a homologous template for accurate repair; involves Rad51, Rad52, Rad59.

• Holliday Junction: Cross-shaped DNA structure formed during HR; resolved by specific enzymes.

• Non-Homologous End Joining (NHEJ): Directly ligates broken DNA ends; more error-prone.

Transposons and Alu Sequences

• Transposons: Mobile genetic elements; can be replicative (copy-and-paste) or conservative (cut-and-paste).

• Alu Sequences: Short, repetitive elements abundant in the human genome.

Chapter 18: Gene Expression I: The Genetic Code and Transcription

Central Dogma of Molecular Biology

• Central Dogma: Information flows from DNA → RNA → Protein.

• Exceptions: Reverse transcription (RNA → DNA), some RNA viruses.

Transcription and Translation

• Transcription: Synthesis of RNA from a DNA template by RNA polymerase.

• Translation: Synthesis of protein from an mRNA template by ribosomes.

Types and Functions of RNA

• mRNA (messenger RNA): Encodes protein sequences.

• rRNA (ribosomal RNA): Structural and catalytic component of ribosomes.

• tRNA (transfer RNA): Brings amino acids to the ribosome during translation.

Prokaryotic vs. Eukaryotic Transcription and Translation

• Promoters: Prokaryotes have -10 (Pribnow box) and -35 sequences; eukaryotes have TATA box, BRE, and enhancers.

• Location: Prokaryotic transcription and translation are coupled in the cytoplasm; eukaryotic transcription occurs in the nucleus, translation in the cytoplasm.

• RNA Polymerases: Prokaryotes have one; eukaryotes have three (I, II, III) with distinct functions.

• Polyribosomes: Multiple ribosomes translating a single mRNA simultaneously.

Reverse Transcriptase

• Function: Synthesizes DNA from an RNA template; used by retroviruses and in molecular biology techniques.

Key Differences Between DNA and RNA

• Sugar: DNA contains deoxyribose; RNA contains ribose.

• Bases: DNA uses thymine; RNA uses uracil.

• Structure: DNA is usually double-stranded; RNA is single-stranded.

Transcription Process

• Initiation: RNA polymerase binds promoter (with sigma factor in bacteria or transcription factors in eukaryotes).

• Elongation: RNA polymerase synthesizes RNA in the 5' to 3' direction.

• Termination: Rho-dependent or intrinsic (hairpin) in bacteria; polyadenylation signal in eukaryotes.

Promoter Elements and Transcription Factors

• TATA Box: Core promoter element in eukaryotes.

• BRE: B recognition element; binds transcription factors.

• Enhancer Elements: Increase transcription from a distance.

• Transcription Factors: Proteins that regulate RNA polymerase binding and activity.

RNA Processing in Eukaryotes

• Polyadenylation: Addition of a poly(A) tail to mRNA 3' end.

• Introns and Exons: Introns are non-coding regions removed by splicing; exons are coding sequences.

• RNA Splicing: Removal of introns by spliceosomes or self-splicing introns.

• mRNA Structure: 5' cap, coding region, 3' poly(A) tail.

Ribosomal RNA Subunits

Other RNA Types and Nuclear Structures

• snoRNAs: Small nucleolar RNAs; guide chemical modifications of rRNA.

• hnRNA: Heterogeneous nuclear RNA; precursor to mRNA.

• pre-mRNA: Primary transcript before processing.

• snRNA: Small nuclear RNA; components of spliceosomes.

• PML Bodies: Nuclear structures involved in gene regulation.

Chapter 19: Gene Expression II: Protein Synthesis and Sorting

The Genetic Code

• Triplet Code: Three-nucleotide codons specify amino acids.

• Start Codon: AUG (methionine in eukaryotes, formylmethionine in prokaryotes).

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

Mutations and Their Effects

• Sickle Cell Anemia: Caused by a missense mutation (Glu → Val) in the β-globin gene.

• Frameshift Mutations: Insertions or deletions that alter the reading frame.

• Missense Mutation: Changes one amino acid.

• Nonsense Mutation: Introduces a premature stop codon.

• Silent Mutation: No change in amino acid sequence.

• Nonstop Mutation: Loss of stop codon, leading to extended translation.

• Large Scale Mutations: Affect large DNA segments (e.g., deletions, duplications, inversions).

Genes and Their Definition

• Gene: DNA sequence encoding a functional product (protein or RNA).

Ribosomes and Translation

• Ribosome Sites: A (aminoacyl), P (peptidyl), E (exit) sites coordinate tRNA binding and peptide synthesis.

• Translation Mechanism: Initiation (assembly of ribosome on mRNA), elongation (peptide bond formation), termination (release of polypeptide).

• Shine-Dalgarno Sequence: Prokaryotic ribosome binding site on mRNA.

• Kozak Sequence: Eukaryotic consensus sequence for translation initiation.

tRNA and Aminoacyl-tRNA Synthetase

• tRNA: Adaptor molecule with anticodon and amino acid attachment site.

• Aminoacyl-tRNA Synthetase: Enzyme that attaches amino acids to their corresponding tRNAs.

• Anticodon: Three-nucleotide sequence on tRNA complementary to mRNA codon.

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

Prokaryotic vs. Eukaryotic mRNA

• Prokaryotic mRNA: Often polycistronic (encodes multiple proteins), no 5' cap or poly(A) tail.

• Eukaryotic mRNA: Monocistronic, has 5' cap and 3' poly(A) tail.

Molecular Chaperones and Protein Folding

• Molecular Chaperones: Assist in proper protein folding; prevent aggregation.

• GroEL/GroES Complex: Bacterial chaperonin system that facilitates folding of newly synthesized proteins.

• Protein Folding Mechanism: Proteins fold into their native conformation, sometimes requiring chaperones.

Suppressor tRNAs

• Function: tRNAs that recognize stop codons and insert an amino acid, suppressing nonsense mutations.

Posttranslational Modification

• Definition: Chemical modifications after translation (e.g., phosphorylation, glycosylation, cleavage).

• Example: Insulin is synthesized as preproinsulin, then processed to active insulin by cleavage of signal and connecting peptides.