Nucleus, chromosomes, DNA- Extended explanation

Eukaryotic DNA is packaged into chromatin, which exists in two primary forms based on compaction, gene accessibility, and function

1. Nucleus, chromosomes, DNA- Part 2 Dr. Vividha Raunekar

2. Heterochromatin and Euchromatin Eukaryotic DNA is packaged into chromatin , which exists in two primary forms based on compaction, gene accessibility, and function: Euchromatin (transcriptionally active, loosely packed). Heterochromatin (transcriptionally silent, highly compacted). 1. Euchromatin: The Transcriptionally Active Chromatin Structure and Composition Euchromatin is a less condensed, lightly stained form of chromatin visible under a microscope. It comprises actively transcribed genes , where RNA polymerase and transcription factors have access to the DNA . Histone Modifications in Euchromatin: High acetylation levels : H3K9ac, H3K14ac reduce nucleosome-DNA interaction, making DNA more accessible. Low DNA methylation : Less CpG methylation enables gene expression.

3. Functions Gene Expression Regulation: Euchromatin is transcriptionally active, allowing for mRNA synthesis. Facilitates DNA Replication: Euchromatin regions replicate early in S phase . Enables Cellular Differentiation: Active euchromatic regions determine cell identity during development. Examples of Euchromatin: Housekeeping Genes: Genes involved in essential cellular functions (e.g., GAPDH, ACTB) are always euchromatic. Active X Chromosome in Females: One X chromosome remains euchromatic, while the other is inactivated as a Barr body (heterochromatin).

4. 2. Heterochromatin: The Transcriptionally Inactive Chromatin Structure and Composition Highly condensed and darkly stained under a microscope. Characterized by low levels of acetylation and high levels of methylation . Organized into heterochromatin domains , often found near centromeres and telomeres . Types of Heterochromatin Constitutive Heterochromatin (always condensed): Found in centromeres, telomeres, and repetitive DNA regions . Highly methylated (e.g., H3K9me3 modification) and resistant to transcription. Example: Satellite DNA in centromeres. Facultative Heterochromatin (can switch between euchromatic and heterochromatic states): Example: X-chromosome inactivation (Barr body) in female mammals. Functions of Heterochromatin Genomic Stability: Prevents transposon activation and genomic instability. Gene Silencing: Restricts access to transcriptional machinery. Protects Chromosome Integrity: Ensures centromere and telomere function .

5. Nucleolus and Ribosome Synthesis The nucleolus is a specialized nuclear subcompartment responsible for ribosome biogenesis , essential for protein synthesis. 1. Structure of the Nucleolus The nucleolus is composed of three distinct regions: Fibrillar Center (FC): Contains rDNA genes and is the site of rRNA transcription by RNA Polymerase I. Dense Fibrillar Component (DFC): Initial processing and modification of rRNA (contains fibrillarin). Granular Component (GC): Final ribosomal assembly before export to the cytoplasm.

6. 2. Ribosome Biogenesis Process Transcription of rRNA (in the FC of the nucleolus) RNA Polymerase I transcribes the 45S precursor rRNA (pre-rRNA). This precursor is processed into three mature rRNAs: 18S, 5.8S, and 28S rRNA . 5S rRNA is transcribed separately by RNA Polymerase III in the nucleoplasm. Processing and Modification of rRNA (in the DFC) Small nucleolar RNAs ( snoRNAs ) guide methylation and pseudouridylation of rRNA. Cleavage of 45S rRNA produces the mature 18S, 5.8S, and 28S rRNAs. Assembly of Ribosomal Subunits (in the GC) 40S subunit (small subunit): Contains 18S rRNA and ribosomal proteins. 60S subunit (large subunit): Contains 28S, 5.8S, and 5S rRNAs, plus ribosomal proteins. Subunits are transported through nuclear pores to the cytoplasm for final assembly into functional ribosomes.

7. 3. Role of the Nucleolus in Cellular Processes Ribosome Biogenesis: Ensures continuous production of ribosomes for protein synthesis. Cell Cycle Regulation: The nucleolus interacts with tumor suppressors ( p53 ) and regulates cell proliferation. Viral Replication Site: Some viruses hijack the nucleolus to enhance replication. Example: Cancer and Nucleolar Hypertrophy Cancer cells exhibit enlarged nucleoli due to high ribosome production, supporting rapid growth. Nucleolar stress responses play a key role in apoptosis and cell survival.

8. Telomeres: Structure, Function, and Aging 1. Telomere Structure Located at the ends of linear chromosomes , telomeres consist of repetitive DNA sequences (TTAGGG in vertebrates) . Protected by the shelterin complex , which includes TRF1, TRF2, POT1, TPP1, TIN2, and RAP1. Forms a T-loop structure that prevents DNA degradation and end-to-end fusion. 2. Functions of Telomeres Prevents DNA Loss: Telomeres protect coding sequences from degradation during replication. Ensures Chromosomal Stability: Prevents chromosome end fusion and rearrangements. Regulates Cell Lifespan: Acts as a mitotic clock for cellular aging.

9. 3. Telomere Shortening and Cellular Senescence Due to the end-replication problem , telomeres shorten with each cell division. When telomeres reach a critical length, cells enter replicative senescence ( Hayflick limit ). 4. Telomerase: The Enzyme that Extends Telomeres Telomerase is a ribonucleoprotein enzyme that adds TTAGGG repeats to chromosome ends. Composed of: TERT (telomerase reverse transcriptase, catalytic subunit). TERC (telomerase RNA template). Highly active in: Germ cells (ensures genetic continuity). Stem cells (prolongs cell renewal). Cancer cells (enables immortality).

10. 5. Telomere Dysfunction and Diseases Cancer: 85–90% of human tumors reactivate telomerase for unlimited proliferation. Aging and Progeria: Shortened telomeres lead to premature aging syndromes (e.g., Hutchinson-Gilford Progeria Syndrome). Dyskeratosis Congenita: A telomerase mutation disorder leading to early stem cell exhaustion. 6. Potential Therapeutic Strategies Telomerase Inhibitors: Target cancer cells (e.g., GRN163L). Telomere Lengthening Therapy: Investigated for anti-aging effects.

11. 1. Structure of DNA Deoxyribonucleic acid ( DNA ) is the hereditary material of all known living organisms and many viruses. Its structure and organization allow it to store, transmit, and replicate genetic information efficiently. 1.1 Chemical Composition of DNA DNA is a polymer of nucleotides , each consisting of: Deoxyribose sugar (a five-carbon pentose). Phosphate group (negatively charged, providing structural stability). Nitrogenous base (adenine, thymine, cytosine, or guanine). The nucleotides are linked by phosphodiester bonds between the 3’-OH of one sugar and the 5’-phosphate of the next nucleotide.

12. 1.2 Nitrogenous Bases and Base Pairing Purines (two-ring structure): Adenine (A) and Guanine (G). Pyrimidines (one-ring structure): Thymine (T) and Cytosine (C). Complementary base pairing follows Chargaff’s Rule (A=T, G≡C) : Adenine pairs with Thymine via two hydrogen bonds. Guanine pairs with Cytosine via three hydrogen bonds (stronger). This specific base pairing ensures fidelity in DNA replication and transcription .

13. 2. DNA Double Helix Model Proposed by James Watson and Francis Crick (1953) based on X-ray diffraction data from Rosalind Franklin , the DNA double helix is: 2.1 Features of the Double Helix Right-handed helical structure with two antiparallel strands. Diameter: ~2 nm. One complete turn: ~10.5 base pairs (~3.4 nm). Major groove and minor groove: Regions where proteins interact with DNA. Hydrophobic stacking interactions between base pairs stabilize the helix. 2.2 Forms of DNA B-DNA (Physiological form): Right-handed, 10.5 bp per turn, common in cells. A-DNA (Dehydrated form): Right-handed, 11 bp per turn, observed in certain conditions. Z-DNA (Left-handed, zigzag backbone): Found in transcriptionally active regions.

14. 3. Nucleotides and DNA Organization in Chromatin 3.1 Higher-Order DNA Packaging DNA is highly compacted in eukaryotic cells to fit within the nucleus (~2 meters of DNA in a ~10 µm nucleus). Nucleosome (First level of packaging): DNA wraps around histone octamers (H2A, H2B, H3, H4) forming 10 nm "beads-on-a-string" fibers . H1 histone stabilizes linker DNA between nucleosomes. 30 nm Chromatin Fiber: Nucleosomes further coil into a solenoid or zigzag structure. Higher-Order Chromatin Organization: Euchromatin (active, loosely packed). Heterochromatin (inactive, tightly packed, gene-silenced).

15. 4. DNA Replication DNA replication is a semi-conservative process , meaning each daughter DNA molecule contains one original (parental) strand and one newly synthesized strand. 4.1 Steps in DNA Replication 1. Initiation (Origin Recognition and Unwinding) Begins at specific origins of replication ( OriC in prokaryotes, multiple origins in eukaryotes). Helicase ( DnaB in prokaryotes) unwinds DNA at the replication fork. Single-strand binding proteins (SSBs) stabilize unwound DNA. Topoisomerase (Gyrase in prokaryotes) relieves supercoiling ahead of the fork.

16. 2. Elongation (New Strand Synthesis) Primase ( DnaG in prokaryotes, Pol α in eukaryotes) synthesizes RNA primers. DNA Polymerase III (prokaryotes) or DNA Polymerase δ and ε ( eukaryotes) elongate the new strand in the 5' → 3' direction . Leading Strand: Continuously synthesized. Lagging Strand: Synthesized discontinuously as Okazaki fragments (~1000 bp in prokaryotes, ~100–200 bp in eukaryotes). DNA Polymerase I (prokaryotes) or RNase H (eukaryotes) removes RNA primers and replaces them with DNA. DNA Ligase seals nicks between Okazaki fragments. 3. Termination Prokaryotic Termination: Tus proteins bind to Ter sites to stop helicase activity. Eukaryotic Termination: Telomeres prevent information loss at chromosome ends.

17. 5. DNA Repair Mechanisms DNA is constantly exposed to damage from radiation, chemicals, and replication errors . Cells have evolved several repair mechanisms: 5.1 Direct DNA Repair Photoreactivation: Repairs UV-induced pyrimidine dimers using photolyase (not present in humans). O6-Methylguanine DNA Methyltransferase (MGMT): Removes alkyl groups from guanine. 5.2 Base Excision Repair (BER) Corrects single-base modifications (deamination, oxidation, alkylation). DNA glycosylases remove damaged bases, creating an AP site (apurinic/apyrimidinic site). AP endonuclease and DNA polymerase β ( eukaryotes) or Pol I (prokaryotes) fill the gap.

18. 5.3 Nucleotide Excision Repair (NER) Removes bulky DNA lesions , including UV-induced thymine dimers . UvrABC endonuclease complex (prokaryotes) or XPA-XPG proteins (eukaryotes) excise damaged regions. DNA polymerase fills the gap, and ligase seals the strand. 5.4 Mismatch Repair (MMR) Fixes errors introduced during replication (base mismatches). MutS-MutL-MutH complex (prokaryotes) or MSH2-MSH6 complex (eukaryotes) recognizes mismatches and corrects errors. 5.5 Double-Strand Break (DSB) Repair Homologous Recombination (HR): Error-free repair using the sister chromatid as a template (Rad51 and BRCA1/2 in eukaryotes). Non-Homologous End Joining (NHEJ): Error-prone repair that directly joins DNA ends (Ku70/Ku80 complex).

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