DNA replication and transcription
Nucleotides, DNA replication, transcription, translation, mutation and DNA repair
Molecular biology for FCPS Part 1: nucleotide synthesis, DNA replication, telomerase, transcription, splicing, translation, mutations and DNA repair. Each step is linked to the drugs that block it and the diseases its defects cause.
One of the most tested topics in past papers
Nucleotides
- Purines (two rings): adenine, guanine. Pyrimidines (one ring): cytosine, thymine (DNA), uracil (RNA).
- A pairs with T by two hydrogen bonds; G pairs with C by three, so GC-rich DNA melts at a higher temperature.
- Purine synthesis is built on PRPP; rate-limiting enzyme glutamine-PRPP amidotransferase; needs glycine, aspartate, glutamine, CO2 and tetrahydrofolate.
- Pyrimidine synthesis makes the ring first and then adds it to PRPP; rate-limiting enzyme carbamoyl phosphate synthetase II (cytoplasm).
| Enzyme | Reaction | Inhibitor |
|---|---|---|
| Dihydrofolate reductase | Regenerates tetrahydrofolate | Methotrexate, trimethoprim, pyrimethamine |
| Thymidylate synthase | dUMP to dTMP (needs methylene-THF) | 5-fluorouracil |
| Ribonucleotide reductase | Ribonucleotides to deoxyribonucleotides | Hydroxyurea |
| Xanthine oxidase | Hypoxanthine to xanthine to uric acid | Allopurinol, febuxostat |
| IMP dehydrogenase | Guanine nucleotide synthesis | Mycophenolate |
- Hereditary orotic aciduria (UMP synthase deficiency): megaloblastic anaemia not corrected by B12 or folate, orotic acid crystals, normal ammonia; treated with uridine.
- Adenosine deaminase deficiency: dATP builds up and poisons lymphocytes, causing severe combined immunodeficiency.
Purine salvage and Lesch–Nyhan syndrome
- HGPRT recycles hypoxanthine and guanine back to nucleotides. In Lesch–Nyhan syndrome (X-linked, so almost only boys) salvage fails; PRPP accumulates, purine synthesis speeds up, and uric acid is overproduced: gout, urate stones, self-mutilation, dystonia and intellectual disability.
- Allopurinol lowers urate and prevents stones and gout, but it does not change the neurological or behavioural features.
- Allopurinol raises levels of azathioprine and 6-mercaptopurine, which xanthine oxidase normally inactivates.
DNA replication
- Replication is semiconservative: each daughter molecule keeps one parental strand. Cells grown in heavy nitrogen then moved for one round into light nitrogen gave DNA all of intermediate density (the classic density-gradient experiment), ruling out conservative replication.
- It starts at origins and always runs 5' to 3'.
| Protein | Role |
|---|---|
| Helicase | Unwinds the double helix |
| Single-strand binding proteins | Keep strands apart |
| Topoisomerases | Relieve supercoiling; bacterial DNA gyrase (topo II) blocked by fluoroquinolones; human topo I by irinotecan, topo II by etoposide and doxorubicin |
| Primase | Lays an RNA primer |
| DNA polymerase III (prokaryotes) | Extends; proofreads with 3' to 5' exonuclease |
| DNA polymerase I | Removes primers and fills gaps |
| Ligase | Seals Okazaki fragments |
- The leading strand is made continuously; the lagging strand in short Okazaki fragments.
- Telomerase is a reverse transcriptase carrying its own RNA template; it adds repeats to chromosome ends that ordinary polymerases cannot finish. Among normal cells it is most active in germ cells, then stem cells; most somatic cells have little, so telomeres shorten with each division. Cancer cells reactivate it.
Transcription and RNA processing
- Eukaryotic RNA polymerases: I makes rRNA, II makes mRNA (blocked by alpha-amanitin from death cap mushrooms, causing liver failure), III makes tRNA and 5S rRNA. Bacterial RNA polymerase is blocked by rifampicin.
- Promoters (TATA box) and enhancers, bound by transcription factors, control initiation.
- Pre-mRNA is processed in the nucleus: a 5' 7-methylguanosine cap, a 3' poly-A tail, and splicing, in which spliceosomes (snRNPs) cut out introns and join exons. Alternative splicing lets one gene make several proteins. Splice-site mutations cause some beta-thalassaemias; anti-Smith antibodies in SLE target snRNPs.
Translation
- The genetic code is triplet, degenerate (most amino acids have several codons), unambiguous and nearly universal. Start codon AUG (methionine); stop codons UAA, UAG, UGA.
- Ribosomes: eukaryotic 80S (60S + 40S); prokaryotic 70S (50S + 30S). Aminoacyl-tRNA synthetases charge each tRNA with its amino acid (ATP); elongation uses GTP.
- Post-translational changes include phosphorylation, glycosylation, hydroxylation of collagen (vitamin C) and ubiquitination. Mannose-6-phosphate tags lysosomal enzymes; its absence causes I-cell disease.
| Target | Drugs or toxins |
|---|---|
| 30S subunit | Aminoglycosides (misreading, bactericidal), tetracyclines (block tRNA binding) |
| 50S subunit | Chloramphenicol (peptidyl transferase), macrolides (translocation), clindamycin, linezolid |
| EF-2 (eukaryotic) | Diphtheria toxin, Pseudomonas exotoxin A (ADP-ribosylation) |
| 60S rRNA | Ricin, Shiga toxin |
Mutations
| Type | Change | Example |
|---|---|---|
| Silent | Same amino acid | Third-base wobble |
| Missense | Different amino acid | Sickle cell: glutamate to valine at position 6 of beta-globin |
| Nonsense | Premature stop codon | Some beta-thalassaemias |
| Frameshift | Insertion or deletion not a multiple of three | Duchenne muscular dystrophy |
| Trinucleotide repeat | Expanding repeat with anticipation | Huntington (CAG), fragile X (CGG), myotonic dystrophy (CTG), Friedreich ataxia (GAA) |
- Carcinogens and radiation cause somatic mutations in the exposed cells; these are not inherited. Germline mutations, such as inherited TP53 in Li–Fraumeni syndrome (autosomal dominant; sarcomas, breast cancer, brain tumours, adrenocortical carcinoma), are present in every cell.
DNA repair
| System | Damage repaired | Disease when defective |
|---|---|---|
| Nucleotide excision | Bulky lesions, UV thymine dimers | Xeroderma pigmentosum: extreme sun sensitivity, early skin cancers |
| Mismatch repair | Replication mismatches | Lynch syndrome: colorectal and endometrial cancer; microsatellite instability |
| Base excision | Deaminated or oxidised bases | MUTYH-associated polyposis |
| Homologous recombination | Double-strand breaks | BRCA1/2 (breast, ovary); PARP inhibitors exploit this |
| Non-homologous end joining and damage sensing | Double-strand breaks | Ataxia telangiectasia (ATM) |
| Crosslink repair, helicases | Crosslinks, replication stress | Fanconi anaemia, Bloom syndrome |
- Inheritance: the classic DNA repair syndromes (xeroderma pigmentosum, ataxia telangiectasia, Fanconi anaemia, Bloom syndrome) are autosomal recessive. Cancer predispositions from repair genes (Lynch, BRCA) are inherited as autosomal dominant susceptibility, because one inherited faulty copy needs only a second somatic hit.
Remember DNA is copied semiconservatively 5' to 3'; introns are spliced out and exons joined before mRNA leaves the nucleus.
Exam trap Allopurinol lowers urate in Lesch–Nyhan syndrome but does not improve the self-mutilation or neurological disease.
Exam trap Xeroderma pigmentosum is nucleotide excision repair; Lynch syndrome is mismatch repair.
How FCPS asks about this
- Normal cells with the highest telomerase activity: germ cells.
- What happens in splicing: introns removed, exons joined.
- All DNA of intermediate density after one round in light nitrogen: semiconservative replication.
- Allopurinol in Lesch–Nyhan syndrome: lowers urate only; neurology unchanged.
- Megaloblastic anaemia unresponsive to B12 and folate with orotic acid crystals: hereditary orotic aciduria.
- Death cap mushroom poisoning: alpha-amanitin blocks RNA polymerase II.
- Mode of inheritance of classic DNA repair syndromes: autosomal recessive.
Test yourself on this topic
5 of the 34 questions in our bank on this topic. Choose an answer to see why every option is right or wrong.
A single base change converts the codon CAG (glutamine) into UAG in a gene's mRNA. What type of mutation is this, and what is its effect?
Answer: Nonsense; truncated protein
Point mutations: silent (synonymous) changes do not alter the amino acid because of code degeneracy; missense mutations substitute a different amino acid (e.g. sickle cell disease, glutamate to valine at position 6 of beta-globin); nonsense mutations create a stop codon (UAA, UAG, UGA), producing a truncated, usually non-functional protein, and the mRNA is often degraded by nonsense-mediated decay (some beta-thalassaemias). Insertions or deletions not in multiples of three cause frameshifts, altering every downstream codon (Duchenne muscular dystrophy), while in-frame deletions shorten the protein (Becker dystrophy, cystic fibrosis ΔF508). Splice-site mutations alter exon–intron processing.
Mutation types
- Silent: same amino acid
- Missense: different amino acid (sickle cell)
- Nonsense: stop codon (UAA, UAG, UGA) → truncated
- Frameshift: insertion/deletion not multiple of 3 (Duchenne)
- In-frame deletion: Becker, CF ΔF508
Remember Nonsense makes the message stop too soon.
Why the other options are wrong
- Missense; one substitution No amino acid substitution; a stop codon forms.
- Silent; no change The amino acid changes to a stop.
- Frameshift; downstream change A substitution does not shift the frame.
- Splice-site; intron retained Not correct.
A boy with Lesch–Nyhan syndrome is started on allopurinol. What will it achieve?
Answer: Lower urate only; neurology unchanged
Lesch–Nyhan syndrome is X-linked recessive deficiency of HGPRT, the purine salvage enzyme. Unsalvaged purines are turned into uric acid, and de novo synthesis rises: hyperuricaemia, gout, urate stones, plus dystonia, intellectual disability and self-mutilation. Allopurinol lowers urate and protects the kidneys and joints but does not improve the neurological problems.
Lesch–Nyhan
- HGPRT deficiency, X-linked
- High urate: gout, stones
- Self-mutilation, dystonia
- Allopurinol: urate only
Remember Allopurinol saves the kidneys, not the behaviour.
Exam trap Lesch–Nyhan affects boys almost exclusively (X-linked).
Why the other options are wrong
- Cure the self-injury and dystonia Neurological features are not helped.
- Restore HGPRT enzyme activity It does not replace the enzyme.
- Return purine synthesis to normal It mainly blocks xanthine oxidase.
- Nothing, as uric acid is normal here Urate is very high.
A 6-year-old has a wobbly gait since infancy, dilated blood vessels on the conjunctivae and recurrent sinus and chest infections. IgA is low and AFP is raised. Which gene is affected?
Answer: ATM
Ataxia telangiectasia is autosomal recessive, caused by ATM mutations that impair repair of double-strand DNA breaks. Features: cerebellar ataxia from early childhood, oculocutaneous telangiectasia, IgA (and IgG2) deficiency with sinopulmonary infections, raised AFP, extreme sensitivity to radiation, and a high risk of lymphoma and leukaemia. X-rays and radiotherapy should be minimised.
Ataxia telangiectasia
- Gene: ATM (DNA break repair)
- Ataxia + telangiectasia
- Low IgA, infections
- Raised AFP
- Radiation sensitive, lymphoma risk
Remember Wobbly, red-eyed, low IgA, high AFP.
Exam trap Avoid unnecessary X-rays: they cause DNA damage these patients cannot repair.
Why the other options are wrong
- BTK Bruton agammaglobulinaemia: no ataxia.
- WAS Wiskott–Aldrich: eczema, small platelets.
- CD40 ligand Hyper-IgM syndrome.
- NADPH oxidase Chronic granulomatous disease.
Molybdenum is a cofactor for which enzyme involved in purine breakdown?
Answer: Xanthine oxidase
Molybdenum is part of the molybdenum cofactor (molybdopterin) for xanthine oxidase (hypoxanthine → xanthine → uric acid), sulfite oxidase and aldehyde oxidase. Allopurinol and febuxostat inhibit xanthine oxidase (gout prophylaxis; azathioprine and 6-mercaptopurine doses must be reduced with allopurinol). Molybdenum cofactor deficiency causes seizures and sulfite toxicity in infants. Adenosine deaminase deficiency causes SCID; HGPRT deficiency causes Lesch-Nyhan syndrome.
Purine breakdown
- Hypoxanthine → xanthine → uric acid
- Enzyme: xanthine oxidase (molybdenum)
- Inhibitors: allopurinol, febuxostat
- Reduce azathioprine with allopurinol
Remember Molybdenum makes uric acid.
Why the other options are wrong
- Adenosine deaminase Deficiency causes SCID.
- HGPRT Salvage enzyme; Lesch-Nyhan.
- Ribonucleotide reductase Makes deoxyribonucleotides; hydroxyurea target.
- PRPP synthetase Purine synthesis.
A newborn has hyperammonaemia. Which finding separates ornithine transcarbamylase (OTC) deficiency from carbamoyl phosphate synthetase I deficiency?
Answer: Raised urinary orotic acid in OTC deficiency
OTC (X-linked, the commonest urea cycle defect) combines carbamoyl phosphate with ornithine. When it is blocked, carbamoyl phosphate spills into pyrimidine synthesis, raising orotic acid in urine. In CPS I deficiency, carbamoyl phosphate is not made, so orotic acid is normal. Both give hyperammonaemia with low citrulline and respiratory alkalosis. Hereditary orotic aciduria has high orotic acid without hyperammonaemia.
Urea cycle defects
- OTC: X-linked, commonest
- OTC: orotic acid high
- CPS I: orotic acid normal
- Both: hyperammonaemia, low citrulline
Remember Orotic acid up with ammonia up: OTC.
Exam trap Orotic aciduria without hyperammonaemia suggests UMP synthase deficiency.
Why the other options are wrong
- Low blood urea only in OTC deficiency Urea is low in both.
- Raised citrulline in OTC deficiency Citrulline is low in both.
- Metabolic acidosis in OTC deficiency Hyperammonaemia typically causes respiratory alkalosis.
- Hypoglycaemia in CPS I deficiency Not a distinguishing feature.
Where this sits in the CPSP syllabus
- Paper 1: Biomolecules and metabolism, Nucleotides, DNA replication, transcription, translation and repair
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