Table of Contents
1. Introduction
DNA replication is one of the most fundamental processes in all of biology. It is the mechanism by which a cell duplicates its genetic material before division, ensuring that each daughter cell receives an exact copy of the genome. Given that even a single error in the ~3 billion base pairs of the human genome can have catastrophic consequences, the replication machinery has evolved extraordinary accuracy.
To put this in perspective: the overall error rate of DNA replication in humans must be less than 1 mistake per 3 × 10⁹ base pairs. This remarkable fidelity is not achieved by any single mechanism, but by a multilayered system:
| Layer | Error Rate Achieved |
|---|---|
| Accurate DNA synthesis alone | ~1 error per 10³–10⁴ bases |
| + Proofreading during synthesis | ~1 error per 10⁶–10⁷ bp |
| + Post-replication mismatch repair | ~1 error per 10⁹–10¹⁰ bp |
This lesson covers DNA replication in both prokaryotes (primarily E. coli as a model organism) and eukaryotes, along with the major DNA repair systems.
2. Overview: The Central Dogma
The three major processes in gene expression are:
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DNA Replication – DNA is copied with high fidelity to produce daughter DNA molecules.
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Transcription (RNA Synthesis) – The DNA sequence is read and transcribed into mRNA, the intermediate step in protein expression.
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Translation (Protein Synthesis) – The mRNA sequence is decoded (translated) to produce a protein.
This lesson focuses primarily on DNA Replication and the associated DNA Repair mechanisms.
3. Main Features of DNA Replication
DNA replication has several defining characteristics that apply to both prokaryotes and eukaryotes:
3.1 Semiconservative Replication
Each strand of the parental double helix serves as a template for the synthesis of a new complementary strand. After replication, each daughter molecule consists of one original (parental) strand and one newly synthesized strand. This was definitively proven by the Meselson-Stahl experiment (1957).
Meselson-Stahl Experiment: E. coli were grown in a medium containing heavy nitrogen (¹⁵N) so that all DNA was labeled “heavy.” Bacteria were then transferred to light nitrogen (¹⁴N) medium and allowed to replicate. DNA was extracted and centrifuged in a CsCl density gradient. Results showed:
- After 1st generation: All DNA appeared at an intermediate (hybrid) density → one ¹⁵N strand + one ¹⁴N strand.
- After 2nd generation: Half was hybrid, half was fully light (¹⁴N/¹⁴N).
This ruled out conservative (both old strands stay together) and dispersive (strands mixed) models. Replication is definitively semiconservative.
3.2 Bidirectionality
Replication begins at a specific sequence called the Origin of Replication and proceeds in both directions simultaneously, forming two replication forks that move away from the origin. This was demonstrated by Cairns (1962) using radioactive thymine in E. coli, forming what is called a replication eye (bubble).
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Prokaryotes (E. coli): Single origin of replication, called oriC.
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Eukaryotes: Multiple origins of replication (30,000–50,000), allowing the much larger genome to be fully replicated within the cell cycle timeframe.
3.3 Semidiscontinuous Synthesis
Because DNA polymerase can only synthesize DNA in the 5’→3’ direction, and the two template strands run antiparallel (one 3’→5’, one 5’→3’), replication proceeds differently on each template strand:
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Leading strand: Synthesized continuously in the same direction as the replication fork movement.
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Lagging strand: Synthesized discontinuously as a series of short fragments called Okazaki fragments (150–200 nucleotides in eukaryotes; 1,000–2,000 nt in bacteria), which are later joined together.
3.4 Requires RNA Primers
DNA polymerase cannot initiate a new strand from scratch. It can only extend a pre-existing strand with a free 3’-OH group. Therefore, a short RNA oligonucleotide called a primer (~5–10 nt for leading strand; multiple on lagging strand) must first be synthesized by an enzyme called primase. The RNA primer provides the free 3’-OH that DNA polymerase requires to begin adding DNA nucleotides.
3.5 Uses dNTPs as Substrates
The building blocks for DNA synthesis are deoxyribonucleoside triphosphates (dNTPs): dATP, dTTP, dCTP, dGTP. During polymerization, the 3’-OH of the growing strand attacks the α-phosphate of the incoming dNTP, releasing pyrophosphate (PPi). The subsequent hydrolysis of pyrophosphate (PPi → 2 Pi) by pyrophosphatase drives the reaction thermodynamically forward (ΔG < 0), making polymerization essentially irreversible.
Chemical reaction summary:
(dNMP)n + dNTP → (dNMP)n+1 + PPi
The Mg²⁺ ions act as essential cofactors: one coordinates the 3’-OH of the primer, the other interacts only with the incoming dNTP, stabilizing the transition state.
4. The Machinery of Replication: Key Proteins (E. coli)
The collection of proteins working together at the replication fork is called the replisome (or more specifically, the primosome refers to the helicase-primase complex).
| Protein | Function | Molecular Weight | Copies/Cell |
|---|---|---|---|
| Helicase (DnaB) | Unwinds the double helix at the replication fork | 300 kD | ~20 |
| SSB proteins | Stabilize single-stranded DNA, prevent re-annealing | 75 kD | ~500 |
| DNA Gyrase (Topo II) | Relieves torsional stress (supercoiling) ahead of the fork | 400 kD | ~250 |
| Primase (DnaG) | Synthesizes short RNA primers | 60 kD | ~50 |
| DNA Polymerase III | Main enzyme; elongates new DNA strands | 1,000 kD | ~20 |
| DNA Polymerase I | Removes RNA primers; fills gaps | 103 kD | ~300 |
| DNA Ligase | Seals nicks between Okazaki fragments | 74 kD | ~300 |
4.1 DNA Polymerases in E. coli
Arthur Kornberg and colleagues discovered the first DNA polymerase (Pol I) in E. coli in 1955, a landmark discovery. Five DNA polymerases have been identified in E. coli:
| Polymerase | Gene | Role | 3’→5’ Exonuclease (Proofreading) | 5’→3’ Exonuclease | Processivity |
|---|---|---|---|---|---|
| Pol I | polA | Repair, primer removal | Yes | Yes (nick translation) | Low (3–200 nt) |
| Pol II | polB | DNA repair | Yes | No | ~1,500 nt |
| Pol III | polC (dnaE) | Main replicative polymerase | Yes | No | Very high (~500,000 nt) |
| Pol IV | dinB | Non-standard repair (translesion) | No | No | ~7 nt |
| Pol V | umuC | Non-standard repair (translesion) | No | No | ~6–8 nt |
Processivity = the average number of nucleotides added before the DNA polymerase dissociates from the template. The high processivity of Pol III is essential for efficient genome replication; it is maintained by the β-sliding clamp which encircles DNA and keeps Pol III tethered to the template.
Key Properties of All DNA Polymerases:
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Cannot initiate new strands – requires a primer.
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Synthesizes only 5’→3’.
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Proofreading (3’→5’ exonuclease activity) – removes incorrectly incorporated nucleotides.
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DNA Pol I additionally has 5’→3’ exonuclease activity (nick translation) for removing RNA primers.
4.2 DNA Ligase
DNA ligase seals the nick (single-strand break) between Okazaki fragments by catalyzing the formation of a phosphodiester bond between the 3’-OH of one fragment and the 5’-phosphate of the next. This is an energy-requiring reaction:
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In eukaryotes and archaea: ATP is the energy source.
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In bacteria: NAD⁺ is used.
Mechanism:
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AMP is transferred from ATP/NAD⁺ to a lysine residue in ligase (adenylation of enzyme).
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The enzyme transfers AMP to the 5’-phosphate at the nick, activating it.
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The 3’-OH attacks the activated phosphate, displacing AMP and forming the phosphodiester bond.
5. DNA Replication: Stage I – Initiation
Initiation involves identifying where to start, opening the helix, and preparing the template for polymerase action.
5.1 Recognition of oriC (in E. coli)
The origin of replication in E. coli, oriC, is a specific ~245 bp sequence characterized by:
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Four 9-bp repeats (Mer-9): consensus sequence
TTATCCACA→ binding sites for the DnaA protein. -
Three 13-bp repeats (Mer-13): consensus sequence
GATCTNTTNTTTT→ A-T rich region that melts (opens) easily.
5.2 Step-by-Step Initiation
Step 1: ~8 molecules of DnaA (an ATPase) bind the 9-bp repeats at oriC when loaded with ATP. This coils the DNA into a right-handed helix, facilitating opening of the A-T rich 13-bp repeat regions. The histone-like protein HU stabilizes this structure.
Step 2: The DnaA-ATP complex promotes unwinding of the three 13-mer sequences (rich in A=T, which have only 2 hydrogen bonds and melt more easily than G≡C).
Step 3: DnaC (an ATPase that binds ssDNA and DnaB when loaded with ATP) loads DnaB helicase (a hexameric ring) onto the single-stranded lagging strand template. DnaC-ATP interacts with DnaB, activating it. DnaC then hydrolyzes ATP to ADP and dissociates, leaving DnaB to unwind the replication fork.
How Helicase Works: DnaB is a hexameric ring. One DNA strand passes through the central hole and is threaded through loops of two adjacent subunits. ATP binding and hydrolysis cycles drive conformational changes, pulling the DNA through and forcing strand separation. This requires energy (ATP hydrolysis breaks the hydrogen bonds between bases: ΔG > 0).
Step 4: SSB proteins (Single-Strand Binding Proteins) coat and stabilize the separated single-stranded DNA, preventing re-annealing and protecting the template from nucleases.
Step 5: DNA Gyrase (Topoisomerase II) relieves the torsional stress (positive supercoiling) that accumulates ahead of the moving replication fork. It works by:
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Making a transient double-strand cut in the DNA.
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Passing another DNA segment through the cut.
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Religating the cut, thereby introducing or removing supercoils.
Step 6: Primase (DnaG) associates with DnaB (forming the primosome) and synthesizes a short RNA primer (10–60 nt) using the DNA as a template.
Step 7: When DNA Polymerase III binds to the primed DNA, it signals completion of initiation. The Hda protein (an ATPase) then binds to Pol III and causes DnaA to hydrolyze its ATP to ADP, causing the DnaA-ADP complex to disassemble from oriC. ATP reloading of DnaA takes 20–40 minutes, ensuring that a new round of replication cannot begin at oriC until the previous one is complete.
Step 8: Dam methylase methylates the N6 position of adenine in 5’-GATC-3’ palindrome sequences throughout the chromosome. This has two functions:
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Protection against restriction enzymes.
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Replication control: When GATC sites are fully methylated, DnaA binds effectively. Immediately after replication, GATC sites are hemimethylated (only the parental strand is methylated), which prevents premature re-initiation. Dam methylase acts soon but not immediately after replication, allowing the cell to distinguish the parental (template) strand from the new strand.
6. DNA Replication: Stage II – Elongation
6.1 Leading Strand Synthesis
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Synthesis is continuous in the 5’→3’ direction toward the replication fork.
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A single RNA primer is made at the origin, and DNA Pol III extends it uninterrupted.
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DNA Pol III has a β-sliding clamp (a ring-shaped dimer) that encircles the DNA template, dramatically increasing processivity.
6.2 Lagging Strand Synthesis – Okazaki Fragments
Because the lagging strand template runs 3’→5’ (opposite to the direction of fork movement), it must be synthesized discontinuously:
a) At regular intervals (~every 1,000–2,000 nt in bacteria), primase (DnaG) associates with DnaB helicase and synthesizes a new short RNA primer.
b) DNA Pol III binds to the primer (loaded onto a β-clamp by the clamp loader complex) and synthesizes a new Okazaki fragment in the 5’→3’ direction (away from the fork), until it reaches the 5’ end of the previously synthesized Okazaki fragment.
c) When synthesis of one Okazaki fragment is complete, the β-sliding clamp detaches. The DNA Pol III core subunits transfer to a new β-clamp that has been loaded on the next primer, beginning synthesis of the next Okazaki fragment.
Key insight – “trombone model”: The lagging strand template loops back through the replisome so that both leading and lagging strand synthesis can occur simultaneously in the same overall direction. The loop grows as a new Okazaki fragment is being synthesized, then it releases and reforms. This physical model explains how a single replisome can coordinate both strands.
d) Each DNA Pol III holoenzyme has 3 core subunits that allow it to simultaneously synthesize 1 or 2 Okazaki fragments while remaining attached to the leading strand template.
6.3 Primer Removal and Gap Filling
After Okazaki fragments are synthesized:
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DNA Polymerase I uses its 5’→3’ exonuclease (nick translation) activity to remove the RNA primer while simultaneously filling in the gap with DNA nucleotides.
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DNA Ligase then seals the remaining nick (phosphodiester bond between the 3’-OH and 5’-phosphate), using energy from ATP (or NAD⁺).
6.4 Fidelity of Elongation
The geometry of correct Watson-Crick base pairs (A=T and G≡C) is nearly identical, and this geometric constraint contributes to the fidelity of replication: incorrect base pairs cannot fit properly into the active site of DNA polymerase.
Proofreading (3’→5’ exonuclease): If an incorrect nucleotide is incorporated:
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The polymerase stalls (cannot translocate with a mismatch at the insertion site).
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The 3’ end of the growing strand is repositioned into the 3’→5’ exonuclease site.
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The incorrect nucleotide is hydrolyzed off (dCMP is released, for example).
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The 3’ end returns to the polymerase active site.
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The correct nucleotide is incorporated.
This reduces the base-pairing error rate from ~1/10³–10⁴ to ~1/10⁶–10⁸.
7. DNA Replication: Stage III – Termination
7.1 Termination in E. coli
The two replication forks travel in opposite directions from oriC around the circular chromosome until they meet at a terminal region on the opposite side.
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This region contains multiple copies (5 on each side) of a 20-bp sequence called Ter.
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The protein Tus (Terminus Utilization Substance) binds to Ter sites, forming a Tus-Ter complex that acts as a polar roadblock: it stops a replication fork approaching from one direction but not the other.
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The Ter sites are arranged so that they create a “fork trap” – forks can enter the region but cannot exit. Only one Tus-Ter complex is used per replication cycle.
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When the two forks converge, the remaining ssDNA is filled in and the strands are ligated.
7.2 Decatenation
After replication of the circular chromosome is complete, the two daughter chromosomes are interlocked (catenated) – they are topologically linked like the rings of a chain. Topoisomerase IV (a type II topoisomerase in E. coli) resolves these catenanes by transiently cutting both strands of one DNA molecule and passing the other through, then re-ligating, until the two chromosomes are completely separated.
8. DNA Repair Systems
DNA molecules are irreplaceable – unlike damaged proteins or RNA which can simply be re-synthesized from the DNA template, damage to DNA itself must be repaired. An irreversible change in DNA sequence is called a mutation.
Types of Mutations:
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Substitution mutation: One base pair replaced by another.
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Insertion mutation: Addition of a base pair.
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Deletion mutation: Removal of a base pair.
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Silent mutation: Occurs in a non-coding region or does not affect gene function.
Types of DNA Damage:
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Replication errors – misincorporated bases.
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Environmental damage – UV radiation (pyrimidine dimers), alkylating agents.
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Oxidative stress (ROS) – formation of 8-oxoguanine (an oxidized form of guanine that mispairs with adenine instead of cytosine, causing G:C → T:A transversions).
Note: ~200 genes in humans encode proteins involved in DNA repair, reflecting the critical importance of genomic integrity. Many repair processes are energetically costly, but the integrity of genetic information takes priority.
8.1 Mismatch Repair (MMR)
Purpose: Corrects base-pairing errors that escape proofreading after replication.
The challenge: How does the repair system know which strand is the “correct” one and which contains the error? In E. coli, the answer lies in Dam methylation. The parental strand is fully methylated at GATC sequences; the newly synthesized strand is not yet methylated (hemimethylated state). The repair system targets the unmethylated (new) strand for correction.
Key proteins in E. coli MMR:
| Protein | Function |
|---|---|
| MutS | Recognizes and binds the mismatch |
| MutL | Adapter; binds both MutS and MutH |
| MutH | Endonuclease; recognizes hemimethylated GATC sequences and cleaves the unmethylated strand |
| DNA Helicase II | Unwinds the DNA at the cleavage site |
| SSB | Stabilizes single-stranded regions |
| Exonucleases (ExoI, ExoVII, RecJ, ExoX) | Degrade the unmethylated strand from the MutH cleavage site to beyond the mismatch |
| DNA Pol III | Re-synthesizes the excised region |
| DNA Ligase | Seals the final nick |
Mechanism:
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MutS detects and binds the mismatch.
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MutL bridges MutS to MutH.
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The MutSLH complex tracks along the DNA to the nearest hemimethylated GATC site.
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MutH cleaves the unmethylated strand on the 5’ side of G in GATC.
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The unmethylated strand is unwound and degraded by exonucleases in the 3’→5’ direction past the mismatch.
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DNA Pol III fills in the gap; DNA Ligase seals the nick.
8.2 Base Excision Repair (BER)
Purpose: Repairs small, chemically altered bases (e.g., deaminated, oxidized, or alkylated bases) that do not significantly distort the DNA helix.
Examples of bases repaired by BER: uracil (deaminated cytosine), hypoxanthine (deaminated adenine), xanthine, 8-oxoguanine, alkylated bases.
Mechanism:
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A DNA glycosylase (there are many, each specific for a type of damaged base) recognizes the lesion and hydrolyzes the N-glycosidic bond, releasing the damaged base and creating an apurinic/apyrimidinic (AP) site.
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An AP endonuclease recognizes the AP site and cuts the DNA backbone (phosphodiester bond) 5’ to the AP site.
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A deoxyribose phosphate lyase removes the remaining deoxyribose-phosphate residue.
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DNA Polymerase I fills in the single-nucleotide gap.
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DNA Ligase seals the nick.
8.3 Nucleotide Excision Repair (NER)
Purpose: Repairs bulky lesions that significantly distort the DNA helix – primarily pyrimidine dimers induced by UV radiation, as well as large chemical adducts (e.g., aflatoxin adducts). Removes and replaces 2–30 nucleotides.
Aflatoxin is produced by molds growing on peanuts and is activated by cytochrome P450 to form a highly reactive compound that covalently modifies DNA bases.
Mechanism (E. coli):
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UvrA recognizes and binds to the damaged site (distortion detector).
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UvrB joins UvrA and helps open the DNA around the lesion.
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UvrC makes two cuts in the damaged strand: one ~8 nt to the 5’ side and one ~4–5 nt to the 3’ side of the lesion, excising an oligonucleotide of ~12–13 nt.
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DNA Helicase II removes the excised oligonucleotide.
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DNA Polymerase I fills the gap (5’→3’), using the intact complementary strand as template.
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DNA Ligase seals the final nick.
In humans, NER defects cause Xeroderma pigmentosum (XP), a disease characterized by extreme sensitivity to UV light and very high rates of skin cancer.
8.4 Direct Repair of UV Photoproducts
UV radiation causes two main types of pyrimidine dimers:
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Cyclobutane pyrimidine dimers (CPDs): Two adjacent pyrimidines (most commonly thymines) are cross-linked through a cyclobutane ring.
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6-4 Photoproducts: A bond forms between C-6 of one pyrimidine and C-4 of the adjacent pyrimidine.
DNA Photolyase (found in bacteria, plants, and non-mammalian species; NOT in placental mammals):
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Binds the distorted DNA in a light-independent step.
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Upon exposure to visible blue light (300–500 nm), it absorbs photons via its cofactor MTHF polyGlu (N⁵,N¹⁰-methenyltetrahydrofolate), which transfers excitation energy to FADH⁻ (flavin adenine dinucleotide, reduced form).
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The excited *FADH⁻ donates an electron to the pyrimidine dimer, generating an unstable dimer radical, which spontaneously breaks the cyclobutane ring.
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The electron is returned to the flavin radical, regenerating FADH⁻ and restoring the original monomeric pyrimidines — no nucleotides are removed or replaced.
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Placental mammals (including humans) lack photolyase and rely on NER to repair UV-induced damage instead.
8.5 Repair of Alkylation Damage (Direct Repair)
Alkylating agents (e.g., nitrosamines, chemotherapy drugs) introduce methyl or ethyl groups onto DNA bases. The most dangerous mutagenic product is O⁶-methylguanine, which mispairs with thymine instead of cytosine, causing G:C → A:T transition mutations.
O⁶-methylguanine-DNA methyltransferase (MGMT):
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Directly transfers the alkyl group from O⁶ of guanine to a cysteine residue in its own active site.
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The alkylated enzyme is permanently inactivated (suicide enzyme) – one molecule of MGMT can only repair a single lesion. This is energetically costly but ensures rapid repair.
9. Eukaryotic DNA Replication
Eukaryotic replication shares the same fundamental principles as prokaryotic replication but has important differences due to the much larger, linear, chromatin-packaged genome.
9.1 Key Differences: Prokaryotes vs. Eukaryotes
| Feature | Prokaryotes (E. coli) | Eukaryotes |
|---|---|---|
| Genome size | ~4.6 Mb | ~3,000 Mb (human) |
| DNA structure | Circular, naked | Linear, wrapped in nucleosomes |
| Origins | Single (oriC) | Multiple (30,000–50,000) |
| Replication time | ~40 min | 6–8 hours (S phase) |
| Main replicative helicase | DnaB (on lagging strand, 5’→3’) | MCM2-7 (on leading strand, 3’→5’) |
| Origin recognition | DnaA protein | ORC (Origin Recognition Complex) |
9.2 The Cell Cycle and Replication Timing
DNA replication occurs during the S phase of the eukaryotic cell cycle:
| Phase | Duration | Events |
|---|---|---|
| G1 | 3–4 h | Growth; pre-replicative complex (pre-RC) assembly |
| S | 6–8 h | DNA synthesis |
| G2 | 1 h | Growth; preparation for mitosis |
| M | 6–12 h | Mitosis |
9.3 Regulation by Cyclins and CDKs
Replication is tightly regulated by cyclins (cell cycle regulatory proteins) and cyclin-dependent kinases (CDKs):
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At the end of M phase, cyclins are ubiquitinated (tagged) and degraded by the proteasome.
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The removal of cyclins in G1 allows the assembly of pre-replicative complexes (pre-RC) at origins.
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Pre-RC components include: ORC (Origin Recognition Complex, analogous to DnaA), Cdc6, and Cdt1.
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When cyclins re-accumulate during S phase, CDKs become active and phosphorylate various proteins, triggering:
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Unwinding of DNA.
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Recruitment of DNA polymerases.
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Initiation of replication.
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After firing, the same origin cannot re-fire in the same cell cycle (inhibition of pre-RC re-formation).
9.4 Eukaryotic Helicases: MCM2-7
In eukaryotes, the main replicative helicase is the MCM2-7 complex (a hexameric ring, analogous to DnaB):
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Loaded onto origins by ORC (analogous to DnaA’s role).
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Moves in the 3’→5’ direction along the leading strand template (opposite to DnaB, which moves 5’→3’ on the lagging strand template).
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Two MCM2-7 hexamers are loaded at each origin (one for each replication fork), ready to fire bidirectionally.
9.5 Eukaryotic DNA Polymerases
| Polymerase | Location | Role |
|---|---|---|
| Pol α | Nucleus | Associates with primase; synthesizes RNA-DNA hybrid primer (~10 RNA + ~20 DNA nt) |
| Pol β | Nucleus | DNA repair (primarily base excision repair, BER) |
| Pol γ | Mitochondria | Mitochondrial DNA replication; has polymerase + exonuclease activity |
| Pol δ | Nucleus | Synthesizes the lagging strand; displaces RNA primer (processed by FEN-1 and RNase H); has 3’→5’ proofreading activity |
| Pol ε | Nucleus | Synthesizes the leading strand; DNA repair after NER; has proofreading activity |
| Pol η, θ, ι | Nucleus | Bypass polymerases – act when DNA is damaged (translesion synthesis) |
10. Telomeres and Telomerase
10.1 The End-Replication Problem
Linear chromosomes pose a unique problem: because RNA primers must be removed and because DNA polymerase can only extend 3’→OH ends, the 5’ end of each newly synthesized strand cannot be fully replicated. When the terminal RNA primer is removed, it leaves a single-stranded 3’ overhang on the parental template strand. This gap cannot be filled in by any conventional DNA polymerase, resulting in chromosome shortening with each division.
Without a solution, chromosomes would lose coding sequences within a few cell divisions. The solution is telomeres and telomerase.
10.2 Telomere Structure
Telomeres are non-coding repetitive DNA sequences at the ends of all linear chromosomes:
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In humans: Thousands of repetitions of 5’-TTGGGG-3’ (G-rich strand) paired with 3’-AACCCC-5’ (C-rich strand), totaling 92 telomeric sequences per human cell (2 per chromosome × 46 chromosomes).
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In S. cerevisiae: G₂.₃(TG)₁–₆ repeats, ~325–400 bp total.
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In plants: 5’-TTTAGGG-3’, 2–9 kb total.
Telomeres terminate in a 3’ single-stranded overhang (G-tail). This single-stranded tail folds back and invades the double-stranded telomere region to form a T-loop structure, which:
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Protects the chromosome end from being recognized as a double-strand break.
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Prevents activation of DNA damage signaling.
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Protects from exonucleases.
The T-loop is maintained by the shelterin complex, which includes:
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TRF1 (Telomeric Repeat binding Factor 1): Binds double-stranded telomeric DNA.
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TRF2: Also binds double-stranded telomeric DNA; critical for T-loop formation and protecting chromosome ends.
10.3 Telomerase: The Reverse Transcriptase
Telomerase is a specialized reverse transcriptase (RNA-dependent DNA polymerase) that solves the end-replication problem by extending the 3’ G-rich overhang.
Components of telomerase:
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TERT (Telomerase Reverse Transcriptase): The catalytic protein subunit.
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TERC (Telomerase RNA Component): The RNA template; in humans contains the sequence 3’-AACCCCAAC-5’, complementary to the telomeric repeat.
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Accessory proteins (Dyskerin, NOP10, NHP2, GAR1): Stabilize the complex and improve activity.
Mechanism of telomere elongation:
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Telomerase binds to the 3’ single-stranded overhang of the telomere (5’-TTGGGG-3’ template strand) via base pairing with its internal RNA template.
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TERT extends the 3’ overhang using the RNA as template, adding DNA nucleotides in the 5’→3’ direction (synthesis of TTGGGG repeats).
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Telomerase slippage: After synthesizing a repeat, telomerase translocates (slips) along to re-position the RNA template for synthesis of the next repeat. This can be repeated multiple times.
-
Telomerase dissociates.
-
Primase (Pol α) synthesizes an RNA primer on the now-extended 3’ overhang.
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DNA Polymerase δ synthesizes the complementary C-rich strand.
-
The RNA primer is removed, leaving the chromosome longer than it was before.
This was awarded the Nobel Prize in Physiology or Medicine 2009 to Elizabeth H. Blackburn, Carol W. Greider, and Jack W. Szostak for discovering “how chromosomes are protected by telomeres and the enzyme telomerase.”
10.4 Telomerase Activity and Cellular Aging
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In somatic cells (differentiated body cells), telomerase activity is switched off after differentiation. Each cell division shortens telomeres by ~50–200 bp.
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After ~50–70 divisions (the Hayflick limit), telomeres become critically short, triggering cellular senescence (permanent cell cycle arrest) or apoptosis (programmed cell death).
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Loss of telomerase activity is a critical molecular mechanism of cellular aging.
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Telomerase remains active in:
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Fetal/embryonic tissue (all cells, enabling rapid growth)
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Germline cells (sperm, eggs – must be immortal across generations)
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Adult stem cells (to maintain stem cell pools)
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The vast majority of cancer cells (~85–90%) – cancer cells “do not age” because continuous telomerase activity prevents telomere shortening, enabling limitless replication (immortalization). This makes telomerase a major target for anticancer drug development.
11. Summary and Exam Highlights
Key Proteins and Their Functions (Quick Reference)
| Protein | Direction | Function |
|---|---|---|
| Topoisomerase II (Gyrase) | — | Introduces/eliminates supercoils; prevents DNA breakage from torsion |
| Helicase (DnaB) | 5’→3’ on lagging strand | Separates the two DNA strands |
| Primase (DnaG) | — | Synthesizes RNA primers (10–60 nt) |
| SSB proteins | — | Prevent re-annealing of separated strands |
| DNA Polymerase III | 5’→3’ synthesis | Synthesizes new DNA strands; has 3’→5’ proofreading |
| DNA Polymerase I | 5’→3’ synthesis + 5’→3’ exo | Removes RNA primers (nick translation); fills gaps |
| DNA Ligase | — | Seals nicks between Okazaki fragments via phosphodiester bonds |
| Telomerase | 5’→3’ (reverse transcriptase) | Extends telomeres using internal RNA template |
The Three-Stage Error Rate Reduction
DNA synthesis alone: 1 error / 10³–10⁴ bases
↓ Proofreading (3'→5' exo)
After proofreading: 1 error / 10⁶–10⁷ bases
↓ Post-replication MMR
After mismatch repair: 1 error / 10⁹–10¹⁰ bases
DNA Repair – Comparison Table
| System | Type of Damage | Key Enzymes | Bases Removed |
|---|---|---|---|
| Mismatch Repair | Replication errors (mismatches) | MutS, MutL, MutH; Pol III; Ligase | Many (up to the GATC site) |
| Base Excision Repair | Small altered bases (uracil, 8-oxoG, alkylated) | DNA glycosylase; AP endonuclease; Pol I; Ligase | 1 base |
| Nucleotide Excision Repair | Bulky adducts, pyrimidine dimers | UvrA, UvrB, UvrC; Pol I; Ligase | 12–13 nt (prokaryotes); 25–30 nt (eukaryotes) |
| Direct Repair (Photolyase) | Cyclobutane pyrimidine dimers (UV) | DNA photolyase + light | 0 (reversal, not excision) |
| Direct Repair (MGMT) | O⁶-methylguanine | MGMT methyltransferase | 0 (methyl group transferred) |
Critical Conceptual Points
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Why can’t DNA polymerase start from scratch? It lacks the ability to position the first nucleotide without a free 3’-OH; a primer provides this.
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Why is the lagging strand synthesized in fragments? Because DNA synthesis must be 5’→3’, but the lagging strand template runs 3’→5’ relative to fork movement, requiring repeated primer synthesis and extension toward the fork.
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Why do cancer cells need telomerase? Without it, their telomeres would shorten with each division (just like normal cells), eventually triggering senescence or apoptosis and halting tumor growth.
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How does the mismatch repair system know which strand to correct? Through the pattern of Dam methylation: the parental strand is methylated at GATC, the new strand is not (hemimethylated). MutH cleaves only the unmethylated strand.
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Why is MGMT called a “suicide enzyme”? Because it irreversibly transfers the methyl group to itself (its cysteine residue), permanently inactivating the enzyme after a single repair event.