DNA Structure and Packaging

DNA Structure

• DNA has a double helix shape
• It consists of two antiparallel strands
• The sugar-phosphate backbone has deoxyribose sugars linked by phosphodiester bonds
• Nitrogenous bases include Adenine (A), Thymine (T), Cytosine (C), and Guanine (G)
• A pairs with T, connected by 2 hydrogen bonds
• C pairs with G, connected by 3 hydrogen bonds
• Major and minor grooves are important for protein binding

DNA Packaging

• DNA is compacted into chromatin to pack into the nucleus

Levels of DNA Packaging

• Nucleosomes involve DNA wrapped around histone proteins
• The 30 nm fiber is nucleosomes coiled into a solenoid structure
• Looped domains are fibers looped onto a scaffold
• Chromosomes are a highly condensed structure found in dividing cells

Nucleosomes

• Core histones (H2A, H2B, H3, H4) form an octamer
• H1 histone links nucleosomes
• Acetylation loosens chromatin and increases transcription
• Methylation can repress or activate gene expression
• Phosphorylation is involved in DNA repair

Cell Cycle Regulation

• The phases include G1, S, G2, M
• Cyclins are regulatory proteins that activate CDKs
• Cyclin-Dependent Kinases (CDKs) drive cell cycle transitions
• p53 is a tumor suppressor that induces apoptosis after DNA damage
• Rb protein regulates the transition from G1 to S phase

DNA Replication

• Involves a Semiconservative Mechanism
• Helicase unwinds DNA
• Topoisomerase relieves supercoiling
• Single-Strand Binding Proteins (SSBs) prevent reannealing
• DNA Polymerase III synthesizes new DNA
• Primase adds RNA primers
• DNA Ligase joins Okazaki fragments
• Telomerase extends telomeres in eukaryotes

DNA Damage

• Point mutations are base substitutions that can be missense, nonsense, or silent
• Frameshift mutations are insertions or deletions
• Thymine dimers are UV-induced damage
• Double-strand breaks are due to radiation or oxidative stress

DNA Repair Mechanisms

• Mismatch Repair (MMR) corrects replication errors
• Base Excision Repair (BER) fixes single damaged bases
• Nucleotide Excision Repair (NER) removes bulky lesions like thymine dimers
• Homologous Recombination (HR) repairs double-strand breaks using a sister chromatid
• Non-Homologous End Joining (NHEJ) joins broken DNA ends, and is error-prone

Central Dogma and Gene Regulation

• The flow of genetic information is DNA → RNA → Protein
• Transcription occurs when RNA polymerase synthesizes mRNA
• Translation occurs when ribosomes translate mRNA into proteins

Eukaryotic Gene Structure

• The promoter is the binding site for RNA polymerase
• Enhancers/Silencers regulate transcription
• Introns are removed via splicing
• Poly-A tail and 5' cap add stability and initiate translation

Regulation of Gene Expression

• Transcription factors bind to DNA to activate/repress transcription
• Enhancers & Silencers increase or decrease gene expression
• Epigenetics uses histone modifications and DNA methylation to control gene activity
• Post-transcriptional regulation involves miRNAs and alternative splicing

Toxicity of DNA Polymerase Inhibitors

• Nucleotide analogs cause chain termination
• Inhibitors block polymerase function
• Effects include cell cycle arrest
• Cells also experience DNA damage accumulation
• Apoptosis can occur in rapidly dividing cells

Chromosome Packing and Condensation

• Human cells package 2 meters of DNA into a small nucleus

Primary Structure

• DNA wraps around histone proteins to form nucleosomes
• Each nucleosome contains 146 base pairs of DNA
• DNA wraps approximately 1.8 times around the histone octamer

Higher-Order Organization

• Nucleosomes form a "beads-on-a-string" structure
• This structure coils into a 30-nanometer chromatin fiber
• Further coiling creates looped domains
• Loop domains organize into chromonema fibers
• Final condensation occurs during mitosis

Histone H1 Defect Consequences

• A genetic defect that prevents histone H1 production would have severe consequences for DNA packaging

Immediate Effects

• Disruption of chromatin fibre formation
• Loss of higher-order chromatin structure
• Reduced chromatin compaction

Cellular impact

• Abnormal gene expression regulation
• Disrupted chromatin territories
• Impaired DNA repair mechanisms
• Potential genomic instability

DNA Replication Stages

• Four stages of replication include initiation, unwinding, elongation, and ligation

Initiation

• Binding of initiator proteins
• Unwinding of double helix
• Primer synthesis

Unwinding

• Helicase separates strands
• Topoisomerase relaxes tension
• Single-strand binding proteins stabilize strands

Elongation

• Synthesis of leading and lagging strands
• RNA primer placement

Ligation

• Okazaki fragment joining
• RNA primer removal
• Final DNA sealing

Replication Statement Errors

• DNA gyrase is a bacterial enzyme, helicase exists in eukaryotic replication
• Primers bind to template strands, are needed for both strands
• DNA polymerase cannot bind directly to template, requires RNA primer for initiation
• DNA ligase joins fragments, not topoisomerase; topoisomerase relaxes supercoiling

Cell Cycle Regulation

• The cell cycle is reliant on interplay between cyclins and cyclin-dependent kinases (Cdks)

Cyclin-Cdk Complexes

• Different cyclins bind specific Cdks
• Complexes form at specific cycle stages
• Activity drives cell cycle progression

Regulation Mechanisms

• Cyclin synthesis and degradation
• Cdk activation and inhibition
• Phosphorylation events
• Feedback control systems

APC/C and Sister Chromatid Separation

• APC/C (Anaphase-Promoting Complex) enables sister chromatid separation

Sister Chromatid Cohesion

• APC/C targets securin for degradation
• Securin normally inhibits separase
• Separase cleaves cohesin proteins

Separation Process

• Cohesin degradation releases sister chromatids
• Chromatids move to opposite poles
• Ensures accurate chromosome distribution

Cell Cycle Checkpoints

• Vital for quality control and cell protection

Quality Control

• Ensures complete DNA replication
• Verifies accurate chromosome alignment
• Checks for DNA damage

Cell Protection

• Prevents damaged cells from dividing
• Allows time for error correction
• Maintains genomic stability

Apoptosis Vs. Necrosis

• Apoptosis is programmed cell death, necrosis is uncontrolled cell death
• Apoptosis causes cell shrinkage and membrane blebbing, and necrosis causes cell swelling and membrane rupture
• Apoptosis doesn't inflame tissues, and necrosis does

Importance of Apoptosis

• Crucial for development and maintenance

Development

• Apoptosis is important for tissue sculpting
• Apoptosis is important for organ formation
• Apoptosis is important for cell number regulation

Maintenance

• Apoptosis is important for removing damaged cells
• Apoptosis is important for preventing cancer
• Apoptosis is important for maintaining tissue homeostasis

P53 Defect Impact

• A p53 defect can affect cell cycle and DNA replication

Cell Cycle

• May result in loss of G1 checkpoint
• May result in uncontrolled cell division
• May result in accumulation of mutations

DNA Replication

• May result in continued replication despite damage
• May result in increased mutation rate
• May result in genomic instability

Ionizing Radiation Damage

• Can cause double-strand breaks
• Can cause base damage
• May cause cross-linking between DNA strands
• May cause chromosomal rearrangements

DNA Repair Mechanism

• Two mechanisms include Non-homologous end joining (NHEJ) and Homologous recombination repair (HRR)

Consequences of Unrepaired DNA

• Unrepaired DNA leads to genetic instability or cellular transformation

Genetic Instability

• Mutations in essential genes
• Chromosomal abnormalities
• Loss of cellular function

Cellular Transformation

• Potential cancer development
• Disrupted cellular regulation
• Uncontrolled cell growth

Telomeres and Aging

• Telomeres contribute to aging through telomere shortening and cellular senescence

Telomere Shortening

• Gradual reduction with each cell division
• Eventual loss of protective function
• Increased risk of chromosomal fusion

Cellular Senescence

• Cells enter dormant state
• Reduced tissue regeneration
• Aging phenotypes occur

Telomerase Activity

• Telomerase maintains telomeres, enables unlimited cell divisions, and contributes to cancer progression
• A Cellular advantage includes preventing telomere shortening, maintaining chromosomal stability and supporting continuous proliferation

DNA Packaging

• The human genome contains ~3 billion base pairs
• The total length is ~2 meters
• DNA is compacted to fit inside the nucleus with a diameter of ~10 µm
• Packaging is important for efficient gene regulation, DNA replication, and protection from damage

Levels of DNA Packaging

• Nucleosomes (first level of compaction) are DNA wrapped around histone proteins
• Each nucleosome contains 147 base pairs of DNA wrapped 1.65 times around a histone octamer
• Histone proteins include H2A, H2B, H3, and H4 (core histones)
• Linker histone H1 binds to the nucleosome and the DNA entry/exit sites to compact it further
• 30 nm chromatin fiber is the second level of compaction, with nucleosomes coiled into thicker fibres, stabilized by histone H1 and histone interactions
• Loop domains are the 3rd level of compaction, with 30nm fibers looped and attached to a non-histone protein scaffold, forming 300 nm loops, anchored to SARs/MARs in the nuclear matrix
• Euchromatin is loosely packed, transcriptionally active, and gene-rich
• Heterochromatin is tightly packed, transcriptionally inactive, and found in centromeres and telomeres
• Chromosome formation is the final level of compaction
• During mitosis, chromatin condenses into visible chromosomes (~700 nm wide) and is controlled by condensins and cohesins

Histone Structure

• Histones are basic proteins, rich in lysine and arginine, to interact with negatively charged DNA phosphate groups
• The histone octamer consists of two copies each of H2A, H2B, H3, and H4; H1 is not part of the octamer, but helps compact nucleosomes

Histone Modifications (Epigenetic Regulation)

• Histones are modified to regulate gene expression
• Acetylation (by HATs) loosens chromatin and activates transcription
• Deacetylation (by HDACs) compacts chromatin and represses transcription
• Methylation (by HMTs) activates or represses transcription
• Phosphorylation & Ubiquitination affects chromatin dynamics and DNA repair

Cell Cycle Phases

• G1 phase (Growth Phase 1) is when the cell grows, increases in size and synthesizes organelles. It checks for suitable DNA replication conditions and commits to division at the Restriction (R) point
• S phase (DNA synthesis) is when DNA replicates forming sister chromatids and histones are synthesized to form chromatin
• G2 phase (Growth Phase 2) prepares cell mitosis, checking for DNA replication errors, and duplicates organelles (e.g., centrosomes)
• M phase (Mitosis & Cytokinesis) is when chromosome segregation and cytoplasm division occur in the cell cycle
• G1 Checkpoint (Restriction Point) checks for DNA damage and sufficient nutrients and is regulated by p53 & Retinoblastoma (Rb) proteins
• G2 Checkpoint - Ensures DNA is fully replicated and undamaged and is controlled by Cyclin B/CDK1 complex
• M Checkpoint (Spindle Assembly Checkpoint): Ensures chromosomes are correctly attached to spindle fibers before anaphase

Regulators

• Cyclins - Regulatory proteins that activate Cyclin-dependent kinases (CDKs)
• CDKs - Enzymes that phosphorylate target proteins to drive the cycle
• p53 halts the cell cycle to trigger apoptosis if repair fails