DNA replication follows the semiconservative model: each parental double helix unwinds, and each strand serves as a template for synthesis of a new complementary strand. The result is two daughter duplexes, each containing one original (parental) strand and one newly synthesized strand. This was elegantly demonstrated by the Meselson-Stahl experiment using density-gradient centrifugation with 15N-labelled DNA.
The mode of DNA replication in which each daughter duplex consists of one original parental strand and one newly synthesized complementary strand.
DNA polymerases can only synthesize DNA in the 5′→3′ direction and require a pre-existing primer (a short RNA or DNA oligonucleotide) to begin synthesis. They catalyse the addition of deoxyribonucleotides to the 3′-OH end of the growing chain, using the template strand to specify each incoming base via Watson-Crick pairing.
Replication begins at specific sequences called origins of replication. In bacteria, a single origin (oriC) suffices for the circular chromosome. In eukaryotes, thousands of origins fire at different times during S phase, allowing the larger genome to be replicated within hours. Origin firing is tightly regulated: origins are licensed by the loading of MCM helicase complexes during G1, and firing is triggered by CDK and DDK kinases at the G1/S transition.
A specific DNA sequence where replication initiates; eukaryotes possess thousands of origins distributed across all chromosomes to complete replication efficiently.
Once an origin fires, the MCM helicase unwinds the DNA, creating a replication bubble. Two replication forks move outward in opposite directions from each origin (bidirectional replication), each fork being a site of active DNA synthesis.
At each replication fork, several enzymes coordinate DNA synthesis. Helicase (MCM complex in eukaryotes) unwinds the parental double helix. Topoisomerases relieve the torsional stress (supercoiling) created ahead of the fork. Single-strand DNA-binding (SSB) proteins stabilize the unwound template strands. Primase synthesizes short RNA primers to provide the 3′-OH needed by DNA polymerase.
The main replicative DNA polymerases in eukaryotes; they synthesize DNA with high processivity (aided by PCNA) and proofread errors via their 3′→5′ exonuclease activity.
Because both template strands are replicated simultaneously but synthesis can only proceed 5′→3′, the two new strands are synthesized differently. The leading strand is synthesized continuously in the same direction as fork movement. The lagging strand is synthesized discontinuously as a series of Okazaki fragments, each beginning with a new RNA primer. RNase H and FEN1 remove RNA primers; DNA polymerase fills the gaps; DNA ligase seals the nicks.
Replicative DNA polymerases achieve high processivity by associating with a sliding clamp (PCNA in eukaryotes), a ring-shaped protein that encircles the DNA and tethers the polymerase to the template. PCNA is loaded onto DNA by the clamp loader (RFC complex), preventing the polymerase from dissociating after each nucleotide addition.
DNA polymerases also have an intrinsic 3′→5′ exonuclease (proofreading) activity. If an incorrect nucleotide is incorporated, the polymerase pauses, excises the mismatched nucleotide, then reinserts the correct one. Proofreading reduces the error rate from ~1 in 105 to ~1 in 107. Post-replication mismatch repair (MMR) further improves accuracy to ~1 in 109.
1. The Meselson-Stahl experiment demonstrated that DNA replication is:
2. Why do DNA polymerases require an RNA primer?
3. Which protein complex acts as a sliding clamp during eukaryotic DNA replication, enhancing polymerase processivity?
4. Okazaki fragments are synthesised on the lagging strand because:
5. The proofreading activity of DNA polymerase uses its:
6. Which enzyme relieves the torsional stress (positive supercoiling) that builds up ahead of the replication fork?
7. In eukaryotes, what regulates the timing of origin firing to prevent re-replication?
8. DNA ligase seals nicks in the DNA backbone by forming:
9. The enzyme that synthesises the short RNA primers needed by DNA polymerase is called:
10. What is the overall direction of synthesis of the lagging strand relative to the direction of replication fork movement?
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With the replication machinery established, we now examine how cells detect and repair the inevitable errors and damage that arise in DNA, and how homologous recombination allows damaged DNA to be repaired using a sister chromatid as template.
DNA is damaged by endogenous sources (reactive oxygen species, spontaneous hydrolysis, replication errors) and exogenous agents (UV light, ionising radiation, chemical mutagens). Common forms of damage include: base depurination (loss of a purine base), base deamination (e.g., cytosine → uracil), pyrimidine dimers (covalent crosslinks between adjacent thymines caused by UV), and double-strand breaks (DSBs) (both strands cut, caused by ionising radiation or stalled replication forks).
A lesion in which both strands of the DNA duplex are severed at or near the same site; the most dangerous form of DNA damage because the chromosome is physically broken.
Base excision repair (BER) corrects small base lesions (oxidised, deaminated, or alkylated bases). A DNA glycosylase removes the damaged base; AP endonuclease cuts the DNA backbone; polymerase and ligase fill and seal the gap.
Nucleotide excision repair (NER) removes bulky helix-distorting lesions such as pyrimidine dimers. A multi-protein complex recognises the distortion, makes incisions ~25 nt on either side of the lesion, excises the damaged oligonucleotide, and fills/seals the gap. Mutations in NER genes cause xeroderma pigmentosum (XP), a disease of extreme UV sensitivity and high skin cancer risk.
Mismatch repair (MMR) corrects base-pair mismatches and small insertions/deletions escaping polymerase proofreading. The MutS homologues (MSH2/6) recognise mismatches; MutL homologues (MLH1/PMS2) coordinate excision and re-synthesis. Defects in MMR cause Lynch syndrome and microsatellite instability in tumours.
DSBs are repaired by two main pathways. Non-homologous end joining (NHEJ) rejoins the broken ends directly with minimal processing. It is fast but error-prone (may lose nucleotides). Homologous recombination (HR) uses a homologous DNA sequence (usually the sister chromatid) as a template for accurate repair. HR requires end-processing by the MRN complex + CtIP to generate 3′ single-strand overhangs, followed by invasion of the homologous duplex by RAD51-coated filaments and new DNA synthesis.
HR is most active in S/G2 phase when sister chromatids are available. NHEJ operates throughout the cell cycle. ATM and ATR kinases are master regulators of the DNA damage checkpoint, signalling DNA damage to halt cell-cycle progression until repair is complete.
Homologous recombination also occurs during meiosis, where programmed DSBs (made by Spo11) initiate crossovers between homologous chromosomes. Crossovers ensure accurate chromosome segregation and create new combinations of alleles (genetic shuffling). The same molecular machinery — strand invasion, synthesis, and resolution — underlies both repair HR and meiotic recombination.
1. Xeroderma pigmentosum (XP) results from defects in which DNA repair pathway?
2. Which repair pathway is preferred in S/G2 phase and uses a sister chromatid as template?
3. Lynch syndrome (hereditary non-polyposis colorectal cancer) is caused by mutations in:
4. Deamination of cytosine produces uracil, which pairs with adenine. If not repaired before replication, this causes a:
5. In base excision repair (BER), the first enzymatic step is:
6. RAD51 is the central recombinase in homologous recombination. Its prokaryotic homologue is:
7. Programmed double-strand breaks during meiosis are introduced by which enzyme?
8. The DNA damage checkpoint is primarily activated by which kinases?
9. NHEJ (non-homologous end joining) differs from HR in that NHEJ:
10. Which type of DNA damage is primarily caused by UV radiation and repaired by NER?
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