DNA Structure and Packaging

DNA Double Helix

The structure of DNA is fundamental to its function in the cell. The most common form is B-form DNA, which is a right-handed double helix.

• Antiparallel Strands: DNA strands run in opposite directions (5' to 3' and 3' to 5').

• Base Pairing: Adenine pairs with Thymine, and Guanine pairs with Cytosine via hydrogen bonds.

DNA Packaging

To fit within the nucleus, DNA is highly organized and compacted.

• Nucleosome: DNA wraps around histone proteins, forming nucleosomes.

• Chromatin Fiber: Nucleosomes are further packed into chromatin fibers.

• Loops: Chromatin fibers form loops anchored to a scaffold.

• Heterochromatin vs. Euchromatin: Heterochromatin is tightly packed and transcriptionally inactive; euchromatin is loosely packed and active.

• Centromeres and Telomeres: Specialized chromosomal regions important for stability and segregation.

Nucleus Structure and Transport

The nucleus is surrounded by a double membrane and regulates transport of molecules.

• Double Membrane: Consists of inner and outer nuclear membranes.

• Nuclear Transport: Proteins with Nuclear Localization Signals (NLS) are imported via importin; Nuclear Export Signals (NES) are exported via exportin.

• Energy Source: Transport is powered by Ran/GTP.

DNA Replication

Semiconservative Replication

Each new DNA molecule contains one old and one new strand.

• Origin of Replication: Specific sequence where replication begins.

• Replication Fork/Bubble: Structure formed during DNA unwinding.

Replication Machinery

• Initiation: DnaA, DnaB, DnaC proteins and Single-Stranded Binding proteins (SSB) help start replication.

• Elongation: DNA synthesis proceeds 5' to 3'. Leading strand is continuous; lagging strand is discontinuous, forming Okazaki fragments.

• Proofreading: DNA polymerase has 3' to 5' exonuclease activity for error correction.

• Replisome Components: DNA helicase unwinds DNA; DNA gyrase/topoisomerase relieves supercoiling; SSB stabilizes single strands; primase synthesizes RNA primers; DNA polymerase synthesizes DNA; DNA ligase joins fragments.

• Trombone Model: Describes coordinated synthesis of leading and lagging strands.

• Telomeres and Telomerase: Protect ends of linear DNA from degradation.

Example: In eukaryotes, telomerase extends telomeres to prevent loss of genetic information during replication.

DNA Damage and Repair

Types of DNA Damage

• Spontaneous Mutations: Mispairing, slippage, and chemical damage.

• Mutagen-Induced Damage: Base analogues, base modifications, intercalating agents.

DNA Repair Mechanisms

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

• Nucleotide Excision Repair (NER): Removes bulky lesions.

• Mismatch Repair: Corrects replication errors; distinguishes new strand by methylation state.

• Translesion Synthesis: Allows replication past DNA lesions.

• Double-Strand Break Repair: Includes Nonhomologous End Joining (NHEJ), Synthesis-Dependent Strand Annealing (SDSA), and Homologous Recombination (HR).

Example: Xeroderma pigmentosum is caused by defects in NER, leading to sensitivity to UV light.

Gene Expression: Transcription and RNA Processing

Central Dogma

Information flows from DNA to RNA to protein.

• Transcription: DNA is transcribed to RNA.

• Genetic Code: Triplet codons, unambiguous, degenerate, non-overlapping.

Mechanisms of Transcription

• Transcription Unit: Region of DNA transcribed into RNA.

• Coding/Template Strand: Template strand is used for RNA synthesis.

• Promoter Features: Prokaryotic promoters differ from eukaryotic promoters.

• Elongation: RNA synthesized 5' to 3'; RNA polymerase may backtrack.

• Three RNAPs in Eukaryotes: RNA polymerase I, II, III; Pol II has a C-terminal domain important for regulation.

• Transcription Factors: Required for initiation and regulation.

• Termination: Ends transcription.

RNA Processing

• rRNA: Cleavage of precursor rRNA.

• tRNA: Four types of processing (e.g., cleavage, base modification).

• mRNA: 5'-cap addition, 3'-polyA tail, splicing; often cotranscriptional.

Example: Alternative splicing allows a single gene to produce multiple protein isoforms.

Gene Expression: Protein Synthesis

Protein Synthesis Players

• Ribosomes: Composed of large and small subunits.

• tRNAs: Carry amino acids to ribosome.

• mRNAs: Provide coding sequence.

• Aminoacyl tRNA Synthetases: Attach amino acids to tRNAs.

Protein Synthesis Steps

• Initiation: Assembly of ribosome, mRNA, and initiator tRNA; requires initiation factors.

• Elongation: Amino acids added at A site, peptide bond formation at P site, exit at E site; elongation factors (EF-Tu, EF-Ts, EF-G) assist.

• Termination: Release factors recognize stop codon and release polypeptide.

• Energy Consumption: GTP and ATP are required for initiation, elongation, and termination.

Protein Folding and Processing

• Chaperones: Hsp70 and Hsp60 (GroEL/GroES) assist folding.

• Degradation of Mutant mRNA: Nonsense and non-stop mediated decay.

• Posttranslational Modifications: Includes phosphorylation, glycosylation, etc.

• Organelle Transport: Proteins transported to mitochondria/chloroplast via transit sequences and complexes (TOM/TIM, TOC/TIC); transported in unfolded state stabilized by chaperones.

Example: Proteins destined for mitochondria are imported through the TOM/TIM complexes.

Gene Expression Regulation

Levels of Regulation

Gene expression is regulated at multiple levels:

• Genome: Chromatin decondensation affects accessibility.

• Transcription: Availability of transcription factors.

• RNA Processing and Nuclear Export: Alternative splicing, mRNA export.

• Translation: Initiation factors, mRNA degradation.

• Posttranslation: Modifications, folding, sorting, regulatory protein interactions.

Additional info: The latter three are collectively called posttranscriptional control.

Cell Cycle and Regulation

Phases of the Cell Cycle

• G1 (G0): Cell growth and preparation.

• S: DNA synthesis.

• G2: Preparation for mitosis.

• M: Mitosis and cytokinesis.

Mitosis

• Five Phases: Prophase, prometaphase, metaphase, anaphase, telophase.

• Key Changes: Chromosome condensation, centrosome movement, nuclear envelope breakdown, microtubule dynamics.

• Cytokinesis: Division of cytoplasm; contractile ring composed of actin and myosin.

Meiosis

• Two Divisions: First division separates homologous chromosomes (reductive); second separates sister chromatids.

• Ploidy and DNA Content: Changes in chromosome number and DNA content (C value).

• Homologous Recombination: Exchange of genetic material during meiosis.

Cell Cycle Regulation

• Checkpoints: Ensure proper progression; transition points.

• Cyclin-Dependent Kinases (Cdks) and Cyclins: Regulate cell cycle; activation depends on cyclin availability and phosphorylation/dephosphorylation.

• Mitotic Cdk: Activates Anaphase Promoting Complex (APC), which degrades securin for chromatid separation and marks cyclin for destruction to exit mitosis.

• p53: DNA damage checkpoint; phosphorylated p53 induces cell cycle arrest or apoptosis.

• Apoptosis: Cell death via caspases after death signals or irreparable damage; anti-apoptotic factors (e.g., Bcl-2) promote survival.

Example: Loss of p53 function can lead to uncontrolled cell division and cancer.

Table: Comparison of DNA Repair Mechanisms

Table: Cell Cycle Phases and Key Events

Key Equations

• Semiconservative Replication: (each daughter contains one parent strand)

• Central Dogma:

• Genetic Code: