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LONG ANSWER QUESTIONS (15 Marks each)

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1. De Novo Synthesis of Purine Nucleotides

Overview

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Step 1: Synthesis of PRPP

• PRPP synthetase is activated by inorganic phosphate and inhibited by purine nucleotides (end-product inhibition).
• PRPP is also used in pyrimidine synthesis and salvage pathways.

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Step 2: Formation of 5-Phosphoribosylamine (Committed Step)

• Catalyzed by glutamine:phosphoribosylpyrophosphate amidotransferase (GPAT).
• This is the committed (rate-limiting) step of purine synthesis.
• GPAT is inhibited by AMP and GMP (feedback inhibition); activated by PRPP.

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Step 3: Synthesis of Inosine Monophosphate (IMP) — 9 Steps

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Step 4: Conversion of IMP to AMP and GMP

• IMP → AMP: Aspartate donates its amino group to IMP (via adenylosuccinate), releasing fumarate. GTP is consumed.
• IMP → GMP: IMP is oxidized to XMP (xanthosine monophosphate), then glutamine donates the amino group. ATP is consumed.
• AMP and GMP are then phosphorylated to ADP/ATP and GDP/GTP by kinases.

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Regulation

• PRPP synthetase and GPAT are feedback-inhibited by AMP and GMP.
• High AMP promotes GMP synthesis; high GMP promotes AMP synthesis.

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Salvage Pathway

• Hypoxanthine + PRPP → IMP (catalyzed by HGPRT)
• Adenine + PRPP → AMP (catalyzed by APRT)
• Deficiency of HGPRT causes Lesch-Nyhan syndrome (self-mutilation, hyperuricemia, gout).

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2. Biosynthesis of Pyrimidine Nucleotides

Key Difference from Purines

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Step 1: Synthesis of Carbamoyl Phosphate (Rate-Limiting Step)

Glutamine + CO₂ + 2 ATP → Carbamoyl phosphate

• Located in the cytosol (contrast: CPS I is mitochondrial, for the urea cycle).
• CPS II is inhibited by UTP (end product) and activated by PRPP.

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Step 2: Formation of Carbamoylaspartate

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Step 3: Ring Closure → Dihydroorotate

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Step 4: Oxidation → Orotate

Note: Steps 1–3 are catalyzed by a single trifunctional enzyme called CAD in mammals.

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Step 5: Attachment to Ribose → OMP

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Step 6: Decarboxylation → UMP

• Deficiency → Hereditary orotic aciduria (orotic acid in urine, megaloblastic anemia).

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Step 7: UMP → UTP → CTP

• UMP is phosphorylated to UDP then UTP by kinases.
• CTP synthetase converts UTP → CTP, using glutamine as the nitrogen donor and consuming ATP.
• CTP provides feedback inhibition on CPS II.

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Step 8: Synthesis of dTMP

• dUMP is methylated to dTMP by thymidylate synthase, using N⁵,N¹⁰-methylene-THF as the carbon donor.
• THF is oxidized to DHF in the process.
• 5-Fluorouracil (anticancer drug) inhibits thymidylate synthase (suicide inhibitor).
• Methotrexate inhibits DHF reductase, depleting THF and blocking both purine and pyrimidine synthesis.

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Regulation Summary

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3. Semiconservative Replication of Double-Stranded DNA + Repair Mechanisms

Meselson–Stahl Experiment

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Steps of DNA Replication

1. Initiation at Origins of Replication

• Replication begins at specific sequences called origins of replication (ori).
• In bacteria: single ori (oriC). In humans: thousands of origins fired simultaneously.
• Initiator proteins recognize ori and recruit replication machinery.

2. Unwinding — Helicase

• Helicase unwinds the double helix by breaking hydrogen bonds between base pairs, creating a replication fork (Y-shaped structure).
• Topoisomerase I/II relieves the torsional stress (supercoiling) ahead of the fork.
• Single-strand binding proteins (SSBPs) stabilize the separated strands.

3. RNA Primer Synthesis — Primase

• DNA polymerase cannot initiate synthesis de novo; it requires a free 3'-OH end.
• DNA primase (an RNA polymerase) synthesizes a short RNA primer (~10 nucleotides) complementary to the template.

4. DNA Synthesis — DNA Polymerase III (in prokaryotes)

• DNA Pol III adds deoxyribonucleotides to the 3'-OH of the primer in the 5' → 3' direction.
• Because both strands are antiparallel, the two strands are replicated differently:

Leading Strand

• Runs 3' → 5' (template direction), so synthesis proceeds continuously in the same direction as fork movement.
• Requires only one primer.

Lagging Strand

• Runs 5' → 3' (template direction), so synthesis proceeds discontinuously (opposite to fork movement).
• Multiple RNA primers are laid down.
• DNA is synthesized in short fragments called Okazaki fragments (~1,000–2,000 nucleotides in prokaryotes; 100–200 in eukaryotes).

5. Removal of RNA Primers + Gap Filling — DNA Pol I

• DNA Pol I (in prokaryotes) uses its 5' → 3' exonuclease activity to remove RNA primers and fills the gaps with DNA using its polymerase activity.
• In eukaryotes, RNase H and FEN1 remove primers; DNA Pol δ/ε fill gaps.

6. Ligation

• DNA ligase seals the nicks between adjacent Okazaki fragments by forming phosphodiester bonds (requires NAD⁺ in prokaryotes; ATP in eukaryotes).

7. Proofreading

• DNA Pol III has a 3' → 5' exonuclease (proofreading) activity that excises mismatched nucleotides, reducing the error rate to ~10⁻⁷–10⁻¹⁰ per base pair.

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DNA Repair Mechanisms

1. Mismatch Repair (MMR)

• Corrects replication errors (base mismatches, small insertions/deletions).
• MutS recognizes the mismatch → MutL is recruited → MutH nicks the newly synthesized (unmethylated) strand → exonuclease removes the error → DNA Pol fills the gap → ligase seals.
• In humans: MSH2, MLH1 genes. Defects → Hereditary Non-Polyposis Colorectal Cancer (HNPCC/Lynch syndrome).

2. Base Excision Repair (BER)

• Corrects small chemical base damage (oxidation, deamination, alkylation).
• DNA glycosylase removes the damaged base, creating an AP (apurinic/apyrimidinic) site.
• AP endonuclease cuts the phosphodiester backbone.
• DNA Pol β fills the gap; DNA ligase seals.

3. Nucleotide Excision Repair (NER)

• Corrects bulky lesions (pyrimidine dimers from UV light, chemical adducts).
• Damage is recognized; a 12–24 nucleotide oligomer is excised around the lesion.
• DNA Pol δ/ε fills the gap; DNA ligase seals.
• Defects → Xeroderma pigmentosum (extreme UV sensitivity, skin cancer).

4. Double-Strand Break Repair

• Homologous Recombination (HR): Uses the intact sister chromatid as a template. Accurate, occurs in S/G2 phase.
• Non-Homologous End Joining (NHEJ): Direct ligation of broken ends, error-prone. Main pathway in G1. Uses Ku proteins, DNA-PKcs.
• Defects in HR: BRCA1/BRCA2 → hereditary breast/ovarian cancer.

5. Photoreactivation (Prokaryotes)

• Photolyase uses visible light energy to directly reverse UV-induced pyrimidine dimers. Not present in humans.

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4. Biosynthesis of Proteins (Translation)

Overview

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Components Required

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Step 1: Transcription and mRNA Processing

• A gene is transcribed by RNA Polymerase II into pre-mRNA.
• Pre-mRNA undergoes 5'-capping (7-methylguanosine), splicing (introns removed by spliceosome), and 3'-polyadenylation (poly-A tail added).
• Mature mRNA is exported to the cytoplasm.

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Step 2: Aminoacylation of tRNA ("Charging")

• Each aminoacyl-tRNA synthetase (one per amino acid) covalently attaches an amino acid to its specific tRNA at the 3'-OH end.
• The reaction: Amino acid + tRNA + ATP → Aminoacyl-tRNA + AMP + PPi
• tRNA contains a 3-nucleotide anticodon that base-pairs with the complementary mRNA codon.

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Step 3: Initiation

1. The small ribosomal subunit (40S) associates with eIF4F (cap-binding complex) at the 5'-cap of mRNA.
2. It scans the mRNA until it finds the AUG start codon (Kozak sequence context).
3. Met-tRNAiMet (initiator tRNA) is delivered to the P site by eIF2-GTP.
4. The large subunit (60S) joins to form the 80S initiation complex.
5. GTP is hydrolyzed and initiation factors are released.

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Step 4: Elongation (Ribosome Has Three Sites: A, P, E)

1. Decoding: EF-Tu-GTP delivers the next aminoacyl-tRNA to the A site. Codon–anticodon matching triggers GTP hydrolysis.
2. Peptide bond formation: Peptidyl transferase (rRNA ribozyme activity of 23S/28S rRNA) transfers the polypeptide from the P-site tRNA to the A-site amino acid.
3. Translocation: EF-G (EF-2 in eukaryotes) + GTP moves the ribosome one codon along the mRNA (A→P→E). The deacylated tRNA exits from the E site.

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Step 5: Termination

• When a stop codon (UAA, UAG, UGA) enters the A site, no aminoacyl-tRNA binds.
• Release factors (RF1/RF2 in prokaryotes; eRF1/eRF3 in eukaryotes) bind and trigger hydrolysis of the peptidyl-tRNA bond.
• The completed polypeptide is released.
• The ribosome dissociates into its subunits (with help from RRF in prokaryotes; ABCE1 in eukaryotes).

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Step 6: Post-translational Modifications

• Folding aided by molecular chaperones (e.g., HSP70, HSP90).
• Glycosylation (in ER/Golgi).
• Phosphorylation, acetylation, ubiquitination.
• Signal peptide cleavage for secretory proteins.
• Protein targeting to mitochondria, nucleus, ER, etc.

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Polysomes

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5. Mutation: Definition and Types

Definition

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Classification of Mutations

A. Based on Type of DNA Change

1. Point Mutations (Single Nucleotide Variants / SNVs)

• One base change → different amino acid in the protein.
• Example: Sickle cell anemia — GAG (Glu) → GTG (Val) in β-globin gene.
• ~40% of all disease-causing variants.
• May cause loss of function, gain of function, or no phenotypic effect.

• Converts an amino acid codon → stop codon (UAG, UAA, UGA).
• Results in a premature termination of the polypeptide → truncated, non-functional protein.
• mRNAs with premature stop codons are often degraded by nonsense-mediated mRNA decay (NMD).
• Example: Many cases of β-thalassemia, Duchenne muscular dystrophy.
• ~10% of all disease-causing variants.

• Base change → same amino acid (due to degeneracy of genetic code).
• No change in protein sequence; generally no phenotypic effect.
• Example: CCU → CCC (both code for proline).

• Alters the normal termination codon → allows translation to continue into the 3' UTR until another stop codon is reached → abnormal elongated protein.

2. Splice-Site Mutations

• Affect the conserved sequences at exon–intron boundaries (GT...AG rule).
• Prevent normal splicing → intron retention or exon skipping.
• Can alter the reading frame.
• Cryptic splice sites: Mutations that activate alternative sites, creating abnormal mRNA.
• ~10% of disease variants.

B. Insertions and Deletions (Indels)

3. Frameshift Mutations

• Insertion or deletion of nucleotides not in multiples of 3.
• Shifts the reading frame beyond the mutation → abnormal downstream amino acid sequence → usually a premature stop codon.
• Example: Duchenne muscular dystrophy (large deletion of dystrophin gene).
• ~25% of disease variants.

4. In-Frame Deletions/Insertions

• Loss or gain of nucleotides in multiples of 3.
• Reading frame is preserved but amino acids are added/deleted.
• Example: Becker muscular dystrophy (in-frame deletion of dystrophin).

C. Dynamic Mutations (Trinucleotide Repeat Expansions)

5. Triplet Repeat Expansions

• Unstable repeats that expand with each generation (anticipation).
• Coding region: polyglutamine (CAG) expansions → Huntington disease, spinocerebellar ataxias.
• Non-coding region: CGG expansions → Fragile X syndrome; CTG → Myotonic dystrophy.
• Larger repeats → earlier onset, more severe disease.

D. Structural Variants (Large-Scale Mutations)

6. Deletions

• Loss of a DNA segment (single gene to entire chromosomal region).
• Example: 22q11 deletion → DiGeorge syndrome.

7. Duplications

• A segment of DNA is duplicated.
• Example: Charcot-Marie-Tooth disease type 1A (17p12 duplication).

8. Inversions

• Segment excised and reinserted in reverse orientation.
• If it disrupts a gene, it causes disease.
• Example: Some cases of Hemophilia A.

9. Translocations

• A chromosomal segment moves to another chromosome.
• Reciprocal: segments exchanged between two chromosomes.
• Robertsonian: fusion of two acrocentric chromosomes.
• Example: t(9;22) Philadelphia chromosome → BCR-ABL → Chronic myeloid leukemia.

E. Based on Effect on Function

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SHORT ANSWER QUESTIONS (5 Marks each)

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Short Answer 1: DNA Repair Mechanisms

1. Mismatch Repair (MMR): Corrects replication mismatches. MutS/MSH proteins recognize mismatches; the newly synthesized (unmethylated) strand is nicked and the error region is excised and resynthesized. Deficiency → Lynch syndrome (HNPCC).
2. Base Excision Repair (BER): Corrects small base damage (oxidation, deamination). DNA glycosylase removes the damaged base → AP endonuclease cuts the backbone → DNA Pol β fills the gap → ligase seals.
3. Nucleotide Excision Repair (NER): Corrects bulky adducts and UV-induced pyrimidine dimers (T-T dimers). A ~24-nucleotide oligomer is excised around the lesion; gap is filled and sealed. Deficiency → Xeroderma pigmentosum.
4. Double-Strand Break Repair:
   • Homologous Recombination (HR): Accurate; uses sister chromatid as template. Key proteins: RAD51, BRCA1/2.
   • Non-Homologous End Joining (NHEJ): Error-prone direct ligation. Key proteins: Ku70/80, DNA-PKcs.
5. Direct Repair: Photolyase (prokaryotes, not humans) directly reverses pyrimidine dimers using light energy. O⁶-methylguanine methyltransferase (MGMT) directly removes alkyl groups.

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Short Answer 2: Genetic Code and Its Characteristics

Definition

Characteristics of the Genetic Code

1. Triplet Code: Each codon consists of 3 consecutive nucleotides. 4³ = 64 possible codons for 20 amino acids + stop signals.
2. Degenerate (Redundant): Most amino acids are specified by more than one codon (e.g., leucine has 6 codons). Degeneracy is mostly in the 3rd (wobble) position. This protects against some mutations being harmful.
3. Non-overlapping: Each nucleotide belongs to only one codon. The reading frame is set from the AUG start codon.
4. Comma-free (Continuous): There are no "punctuation" nucleotides between codons; the code is read continuously from the AUG start codon.
5. Universal: With minor exceptions (mitochondria, some protozoa), the same codon specifies the same amino acid in virtually all organisms.
6. Unambiguous: Each codon specifies only one amino acid (though one amino acid may have many codons).
7. Start Codon: AUG (codes for methionine / formyl-methionine in prokaryotes) initiates translation.
8. Stop Codons: UAA, UAG, UGA — do not code for amino acids; terminate translation.
9. Wobble: The 3rd position of the anticodon can pair with more than one base in the codon (Crick's wobble hypothesis), allowing one tRNA to recognize multiple codons.

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Short Answer 3: Leading and Lagging Strands of DNA

Leading Strand

• Template runs 3'→5' in the direction of fork movement.
• New strand is synthesized continuously in the same direction as the replication fork advances.
• Requires only one RNA primer to initiate synthesis.
• Synthesized in a smooth, uninterrupted manner.

Lagging Strand

• Template runs 5'→3' in the direction of fork movement.
• New strand must be synthesized discontinuously, in the direction opposite to fork movement.
• Multiple RNA primers are required.
• DNA is synthesized in short pieces called Okazaki fragments (~100–200 bp in eukaryotes; 1,000–2,000 bp in prokaryotes).
• RNA primers are later removed, gaps are filled by DNA Pol I (prokaryotes), and fragments are joined by DNA ligase.

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Short Answer 4: Nucleotides — Definition and Four Examples

Definition

1. A nitrogenous base (purine: adenine, guanine; or pyrimidine: cytosine, thymine, uracil)
2. A pentose sugar (ribose in RNA; 2'-deoxyribose in DNA)
3. One or more phosphate groups attached at the 5' carbon of the sugar

Four Important Nucleotides

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Short Answer 5: Onion Peel Model of DNA Replication

Key Features:

1. Multiple Origins: Eukaryotic chromosomes are very large and have thousands of replication origins (unlike bacterial chromosomes which have a single origin). This allows replication to complete within the S phase of the cell cycle (~6–8 hours in humans).
2. Replicons: The DNA between two adjacent origins forms a unit called a replicon. Each replicon is replicated bidirectionally from its origin.
3. Replication Bubbles (Eyes): As each origin fires, bidirectional replication forks proceed outward in both directions, forming "bubbles" or "eyes" that are visible in electron microscopy.
4. Sequential Activation: Origins are not all activated simultaneously; they fire at different times during S phase. Early-replicating regions are generally transcriptionally active (euchromatin); late-replicating regions are heterochromatin.
5. Joining of Bubbles: As replication proceeds, adjacent bubbles merge until the entire chromosome is duplicated.

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Short Answer 6: Okazaki Fragments and Gout

Okazaki Fragments

• Prokaryotes: ~1,000–2,000 nucleotides
• Eukaryotes: ~100–200 nucleotides

1. As the replication fork advances, the lagging strand template is exposed in the 5'→3' direction.
2. Primase repeatedly synthesizes short RNA primers on this template.
3. DNA Pol III (prokaryotes) / DNA Pol α/δ (eukaryotes) extends each primer to produce an Okazaki fragment.
4. Each fragment begins with an RNA primer (~10 nt) followed by newly synthesized DNA.
5. DNA Pol I (prokaryotes) / RNase H + FEN1 (eukaryotes) removes RNA primers and fills gaps.
6. DNA ligase seals nicks between fragments using a phosphodiester bond.

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Gout

• Uric acid is the end product of purine catabolism in humans (produced by xanthine oxidase).
• Hyperuricemia results from:
  • Overproduction of uric acid (10%): HGPRT deficiency, PRPP synthetase overactivity, high purine diet, increased cell turnover.
  • Underexcretion (>90%): Renal urate handling defects, thiazide diuretics, lactic acidosis.

• Acute gouty arthritis: Sudden, severe joint pain, redness, warmth (most commonly first metatarsophalangeal joint — "podagra").
• Intercritical gout: Asymptomatic periods between attacks.
• Tophaceous gout: Chronic nodular MSU deposits (tophi) in soft tissues, earlobes, Achilles tendon.
• Uric acid nephrolithiasis: Kidney stones.

• Acute attack: NSAIDs, colchicine, corticosteroids.
• Long-term: Allopurinol or febuxostat (xanthine oxidase inhibitors) reduce uric acid production; probenecid (uricosuric) increases renal excretion.

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Summary of All Answers

Long Answer Questions (15 Marks)

Short Answer Questions (5 Marks)