DNA Replication
and Repair
Master the molecular machinery of semiconservative replication, the enzymes that drive it, and the repair mechanisms that preserve genomic integrity.
MCAT Biological Foundations
What Is DNA Replication?
DNA replication is the biological process by which a cell produces two identical copies of its DNA before cell division. It ensures that each daughter cell receives a complete and accurate copy of the genome.
When
S phase of interphase, before mitosis or meiosis
Why
To transmit genetic info faithfully to daughter cells
Where
Nucleus (eukaryotes) or nucleoid (prokaryotes)
The Semiconservative Model
Origins of Replication and the Replication Fork
Replication begins at specific DNA sequences called origins of replication (ORI). Prokaryotes have a single origin; eukaryotes have multiple origins per chromosome, allowing the large genome to be copied efficiently.
Replication Fork
The Y-shaped structure where the parental DNA unwinds and new strands are synthesized
Bidirectional
Two replication forks move in opposite directions from each origin, forming a replication bubble
Key Enzymes of Replication
1
Helicase: Unwinds the double helix by breaking hydrogen bonds between base pairs at the replication fork.
2
Topoisomerase (Gyrase): Relieves supercoiling tension ahead of the replication fork by cutting and rejoining DNA strands.
3
Primase: Synthesizes a short RNA primer complementary to the template, providing the free \(3'-\text{OH}\) group DNA polymerase needs.
4
\(\text{Pol III}\) (prokaryotes): The main replicative enzyme that adds nucleotides in the \(5' \to 3'\) direction and has \(3' \to 5'\) proofreading exonuclease activity.
5
\(\text{Pol I}\): Removes RNA primers and fills in the gaps with DNA nucleotides.
6
DNA Ligase: Seals the nicks (phosphodiester bonds) between Okazaki fragments on the lagging strand.
Anatomy of the Replication Fork
Leading vs. Lagging Strand
| Leading Strand | Lagging Strand | |
|---|---|---|
| Direction | Toward the fork | Away from the fork |
| Synthesis | Continuous | Discontinuous |
| Primers needed | One | Many (one per fragment) |
| Okazaki fragments | None | Yes |
| Ligase required? | No | Yes (seals fragments) |
| Template Orientation (re: fork) | \(3' \to 5'\) | \(5' \to 3'\) |
Eukaryotic vs. Prokaryotic Replication
| Prokaryotes | Eukaryotes | |
|---|---|---|
| Origins | Single (oriC) | Multiple per chromosome |
| Main polymerase | \(\text{Pol III}\) | \(\text{Pol }\delta\) and \(\text{Pol }\varepsilon\) |
| Speed | ~1000 nt/s | ~50 nt/s |
| Okazaki fragments | 1000-2000 nt | 100-200 nt |
| Chromosome | Circular | Linear |
| Telomere problem? | No | Yes |
The Telomere Problem
Proofreading: the First Line of Defense
DNA polymerase has built-in \(3' \to 5'\) exonuclease (proofreading) activity. As it synthesizes new DNA, it checks each newly added nucleotide. If a mismatch is detected, the enzyme reverses, excises the incorrect nucleotide, and inserts the correct one.
Error Rate (no proofreading)
\(\sim 1\) error per \(10^5\) nucleotides
Error Rate (with proofreading)
\(\sim 1\) error per \(10^7\) nucleotides
After Mismatch Repair
\(\sim 1\) error per \(10^9 - 10^{10}\) nucleotides
DNA Repair Mechanisms
Mismatch Repair in Detail
1
Recognition: MutS protein detects mismatched base pairs in the newly synthesized strand.
2
Strand discrimination: In prokaryotes, the parental strand is methylated (GATC sites by Dam methylase). The unmethylated new strand is identified as the one containing the error.
3
Excision: MutH nicks the unmethylated strand. An exonuclease degrades the error-containing segment.
4
Resynthesis: \(\text{Pol III}\) fills the gap using the parental strand as template, and ligase seals the nick.
BER and NER: Excision Repair
| Base Excision (BER) | Nucleotide Excision (NER) | |
|---|---|---|
| Damage type | Small, non-bulky (oxidized, deaminated bases) | Bulky, helix-distorting (thymine dimers, adducts) |
| First step | DNA glycosylase removes damaged base | Damage recognition complex scans helix |
| Nucleotides removed | 1 base (creates AP site) | ~12-13 nt oligonucleotide patch |
| Key enzyme | AP endonuclease | Dual incision endonucleases |
| Associated disease | Some cancer syndromes (e.g., MAP) | Xeroderma pigmentosum |
How to Approach Replication Problems
1
Identify the strand: Is the question about the leading or lagging strand? Check if synthesis is continuous or requires Okazaki fragments.
2
Apply the \(5' \to 3'\) rule: All DNA synthesis is \(5' \to 3'\). The template is always read \(3' \to 5'\). If a question mentions directionality, anchor your answer here.
3
Match enzyme to function: Helicase unwinds, primase makes RNA primer, Pol III extends, Pol I replaces primers, ligase seals nicks.
4
Check for repair: If the question involves a mutation or disease, connect it to the specific repair pathway (mismatch, BER, or NER).
5
Distinguish prokaryotic vs. eukaryotic: Watch for clues: circular vs. linear chromosomes, single vs. multiple origins, telomerase involvement.
Worked Example: Enzyme Knockout
1
Recall ligase's role: It seals nicks between Okazaki fragments on the lagging strand by forming phosphodiester bonds.
2
Predict the consequence: Without ligase, Okazaki fragments remain unjoined. The lagging strand would be fragmented.
3
The leading strand would be largely unaffected since it is synthesized as a single continuous piece.
Worked Example: Repair Pathway
1
Identify the lesion: Thymine (pyrimidine) dimers are bulky, helix-distorting lesions caused by UV light.
2
Match to pathway: BER handles small base modifications. NER handles bulky lesions like thymine dimers.
3
Connect to disease: Defective NER causes Xeroderma Pigmentosum (XP).
Common Mistakes to Avoid
✕ Confusing Helicase and Topoisomerase
Helicase unwinds the helix by breaking H-bonds. Topoisomerase relieves tension (supercoiling) by cutting and rejoining the backbone.
✕ Saying Pol III "removes primers"
\(\text{Pol I}\) removes RNA primers and fills gaps. \(\text{Pol III}\) is the main replicative polymerase. Mixing them up is a classic trap.
✕ Mixing up BER and NER triggers
BER: small base damage (oxidation, deamination). NER: bulky helix-distorting lesions (thymine dimers). Size of the lesion is the key distinction.
✕ Forgetting the primer requirement
DNA polymerase cannot initiate synthesis de novo. It requires a free \(3'-\text{OH}\) from an RNA primer. This is why primase is essential.
Summary: Enzymes and Repair
| Component | Function | Key Detail |
|---|---|---|
| Helicase | Unwinds dsDNA | Breaks H-bonds at fork |
| Topoisomerase | Relieves supercoiling | Ahead of fork |
| Primase | Synthesizes RNA primer | Provides \(3'-\text{OH}\) |
| \(\text{Pol III / Pol }\delta\text{ / Pol }\varepsilon\) | Main replication | \(5' \to 3'\) synthesis, \(3' \to 5'\) proofreading |
| \(\text{Pol I}\) | Removes primers | Fills gaps with DNA |
| Ligase | Seals nicks | Joins Okazaki fragments |
| Telomerase | Extends telomeres | Reverse transcriptase |
| Mismatch Repair | Fixes replication errors | Lynch syndrome if defective |
| NER | Removes bulky lesions | XP if defective |
Question 1 of 5
During DNA replication, which enzyme is responsible for synthesizing the RNA primer needed to initiate new strand synthesis?
Primase is an RNA polymerase that synthesizes a short RNA primer (~10 nt) complementary to the template strand, providing the free \(3'-\text{OH}\) group required by DNA polymerase. Helicase unwinds DNA, Pol I removes primers, and ligase seals nicks.
Question 2 of 5
A cell line is found to have a mutation in MutS, rendering it non-functional. Which of the following is the most likely consequence?
MutS is the mismatch recognition protein in the mismatch repair (MMR) pathway. Without functional MutS, replication errors (mismatches) are not detected and corrected, leading to a mutator phenotype and increased cancer risk (Lynch syndrome). Thymine dimers are repaired by NER, not MMR.
Question 3 of 5
The Meselson-Stahl experiment demonstrated that DNA replication is semiconservative. After two rounds of replication in \(^{14}\text{N}\) medium, what fraction of the DNA molecules would be entirely light (\(^{14}\text{N}\)-\(^{14}\text{N}\))?
After 1 round: 2 molecules, both hybrid (\(^{15}\text{N}\)-\(^{14}\text{N}\)). After 2 rounds: 4 molecules total, 2 hybrid + 2 light. So \(\frac{2}{4} = \frac{1}{2}\) are entirely light. Each original heavy strand remains paired with one new light strand, while the other copies are fully light.
Question 4 of 5
A patient with Xeroderma Pigmentosum has a defect in which DNA repair pathway?
Xeroderma Pigmentosum (XP) results from defective nucleotide excision repair (NER). NER removes bulky, helix-distorting lesions such as UV-induced thymine dimers. Without NER, these lesions persist and cause mutations leading to skin cancer. MMR defects cause Lynch syndrome, not XP.
Question 5 of 5
Telomerase is best described as a reverse transcriptase because it:
Telomerase carries its own RNA component (TERC) that serves as a template for extending the 3' overhang of telomeric DNA. Because it synthesizes DNA from an RNA template, it functions as a reverse transcriptase. This prevents progressive telomere shortening in stem cells, germ cells, and cancer cells.