DNA Replication and Repair
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Direct answer
Meselson and Stahl proved in 1958, using heavy nitrogen and caesium chloride density gradients, that DNA replicates semi-conservatively — each daughter duplex keeps one parental strand. Replication is bidirectional from origins: helicase unwinds, primase lays RNA primers, polymerase III builds the leading strand continuously and the lagging in Okazaki fragments, polymerase I excises primers, and ligase seals the nicks. Repair pathways then police the genome, and their failures name the exam's famous diseases: xeroderma pigmentosum (nucleotide excision repair), Lynch syndrome (mismatch repair) and ataxia telangiectasia (double-strand break sensing).
What you must remember
- Replication is semi-conservative, bidirectional and 5-prime-to-3-prime; E. coli has one origin (oriC), human chromosomes have thousands, licensed once per cell cycle.
- Prokaryotic cast: DnaA recognises the origin; DnaB helicase unwinds; single-strand binding proteins protect; DNA gyrase (topoisomerase II) relieves supercoils — the target of fluoroquinolones.
- Primase (DnaG) makes the RNA primer; polymerase III holoenzyme is the main replicative enzyme with 3-prime-to-5-prime proofreading and a sliding beta clamp; polymerase I removes primers and fills gaps; ligase seals phosphodiester bonds.
- Okazaki fragments: roughly 1,000–2,000 nucleotides in bacteria, 100–200 in eukaryotes.
- Eukaryotic polymerases: alpha (primase-associated), delta (lagging strand), epsilon (leading strand) and gamma — the sole mitochondrial replicase, a favourite one-liner.
- Telomerase, a reverse transcriptase carrying its own RNA template, lengthens chromosome ends in germline, stem and cancer cells, solving the end-replication problem.
- Repair roster: base excision repair (glycosylase initiates; handles deamination, oxidation) — ties to uracil-DNA glycosylase and 5-methylcytosine hotspots; nucleotide excision repair removes bulky lesions such as UV pyrimidine dimers — defective in xeroderma pigmentosum.
- Mismatch repair (MutS/MutL in bacteria; MSH2/MLH1 in humans) corrects replication slips; germline defects cause Lynch syndrome (hereditary non-polyposis colorectal cancer) with microsatellite instability.
- Double-strand breaks: homologous recombination repair needs BRCA1/BRCA2 (breast–ovarian cancer predisposition); non-homologous end joining (Ku, DNA-PK, ligase IV) is error-prone; ATM kinase senses breaks — mutated in ataxia telangiectasia with radiosensitivity.
- Direct reversal: O6-methylguanine-DNA methyltransferase removes alkyl groups; photolyase (absent in humans) would undo pyrimidine dimers.
- Combined proofreading and repair push the final error rate to about one in a billion to ten billion bases.
Walking the fork and then repairing it
Visualise the growing fork: gyrase relieves positive supercoils ahead, helicase opens the duplex, single-strand binding protein coats the template, and primase drops an RNA primer. On the leading template, polymerase III (epsilon in humans) runs continuously toward the fork; on the lagging side the polymerase keeps hopping back, building each Okazaki fragment from a fresh primer until polymerase I chews out the RNA (its unique 5-prime-to-3-prime exonuclease) and ligase glues the nicks. At chromosome ends, the lagging strand cannot be finished — hence telomeric shortening in somatic cells, the mitotic clock, countered by reactivated telomerase in over 85 per cent of cancers. Then imagine ultraviolet light fusing adjacent thymine bases into a cyclobutane dimer. In a normal person, the nucleotide excision repair complex recognises the helical distortion, excises a 25–30 nucleotide patch, and polymerase refills. In xeroderma pigmentosum, seven complementation-group genes fail: the child freckles by age two, burns after minutes of sun and develops skin cancers at thousands-fold increased rates — strict photoprotection is the only defence. If instead a single mispaired base escapes polymerase proofreading, mismatch repair fixes it within minutes; when MSH2 or MLH1 is defective, microsatellites lengthen and shorten unchecked, and colon and endometrial cancers follow through the Vogelstein multistep sequence.
Where students slip
Three confusions dominate. Students swap the exonuclease directions: only polymerase I has the 5-prime-to-3-prime exonuclease that removes primers, while both I and III proofread 3-prime-to-5-prime — the exam asks exactly this. Second, they attribute xeroderma pigmentosum to mismatch repair; it is nucleotide excision repair, whereas mismatch repair defects give Lynch syndrome — no photosensitivity, but tumours. Third, remember eukaryotic polymerase gamma is mitochondrial: mutations cause mitochondrial DNA depletion syndromes and are the reason some mitochondrial diseases show maternal plus Mendelian patterns.
Frequently asked questions
What did the Meselson–Stahl experiment demonstrate?
Semi-conservative replication. E. coli grown in 15N then switched to 14N showed, after one generation, hybrid-density DNA and, after two, a 1:1 mix of hybrid and light DNA — exactly the semi-conservative prediction.
Which enzyme removes RNA primers and which seals the nick?
DNA polymerase I digests the primer with its 5-prime-to-3-prime exonuclease while filling in with DNA; ligase then seals the final phosphodiester bond, consuming ATP in eukaryotes (NAD+ in bacteria).
Which repair pathway is defective in xeroderma pigmentosum?
Nucleotide excision repair, which removes bulky adducts such as ultraviolet-induced pyrimidine dimers; patients suffer extreme photosensitivity and early skin and mucosal cancers.
Why do BRCA1 and BRCA2 mutations predispose to cancer?
They are needed for homologous recombination repair of DNA double-strand breaks; without them, breaks are mis-repaired by error-prone pathways, accelerating genomic instability in breast and ovarian epithelium.
What is the end-replication problem and how do cancer cells solve it?
The lagging strand cannot copy the extreme chromosome tip, so telomeres shorten each division; cancer cells reactivate telomerase, a reverse transcriptase with a built-in RNA template, achieving replicative immortality.