DNA Repair
This subtopic covers DNA replication and repair — how the replisome copies DNA and how proofreading, mismatch repair, and excision repair pathways fix errors.
This subtopic covers two closely linked processes: how DNA is replicated (the enzymes and steps that copy DNA), and how DNA is repaired when replication or environmental damage introduces errors. Repair only makes sense in the context of replication, so this subtopic walks through both.
Key Takeaways
Replication starts at the origin of replication, unwound by helicase and stabilized by single-stranded DNA-binding proteins; primase lays RNA primers so DNA polymerase can begin synthesis; topoisomerase relieves supercoiling ahead of the fork.
The leading strand is synthesized continuously (Pol ε in eukaryotes); the lagging strand is synthesized discontinuously as Okazaki fragments (Pol α primes, Pol δ extends), sealed by DNA ligase. Eukaryotic primer removal is handled mainly by Pol δ strand-displacement and FEN1, assisted by RNase H2.
Telomerase extends linear chromosome ends in eukaryotes only, using its own RNA template; circular prokaryotic chromosomes don't need this step.
Replication produces two semiconservative daughter molecules, each with one old strand and one new strand.
Oncogenes (mutated, overactive proto-oncogenes) and tumor suppressor genes (like p53 and Rb, which lose their protective function when mutated) are the two gene categories central to cancer development.
Four DNA repair pathways: proofreading and mismatch repair fix replication errors (mismatch repair via MSH2/MLH1, largely during S phase); nucleotide excision repair and base excision repair fix environmental/chemical damage (bulky lesions vs. small base damage, respectively).
DNA Replication: Setting Up the Replisome
Before walking through replication step by step, it helps to meet the key players first.
Origin of Replication and the Replication Fork
Replication begins at a specific site on the DNA called the origin of replication. Prokaryotic cells (like bacteria) have just one origin per chromosome. Eukaryotic cells, with much larger genomes, have multiple origins per chromosome — allowing replication to proceed quickly across long stretches of DNA.
Unwinding: Helicase and Single-Stranded DNA-Binding Proteins
Once replication begins, helicase unwinds the double helix by breaking the hydrogen bonds between complementary bases, separating the two strands and creating a replication fork on either side of the origin.
The newly exposed single strands are unstable and could snap back together, so single-stranded DNA-binding proteins attach to each strand to hold them apart.
Priming: Primase and RNA Primers
DNA polymerase — the enzyme that builds new DNA — can't start a strand from scratch; it can only add nucleotides to an existing strand. That's where primase comes in, laying down short RNA primers that give DNA polymerase a starting point.
DNA synthesis itself is carried out by different polymerases depending on the organism: primarily DNA polymerase III in prokaryotes, and multiple polymerases in eukaryotes — most notably DNA polymerase alpha, delta, and epsilon.
Relieving Tension: Topoisomerase
As helicase continues unwinding DNA, it introduces positive supercoils ahead of the replication fork, making the DNA tighter and harder to unwind. DNA topoisomerase (called DNA gyrase in prokaryotes) relieves this tension by cutting the DNA, allowing it to unwind, and resealing it.
Leading vs. Lagging Strand Synthesis
At each replication fork, the two template strands run in opposite directions. Because DNA polymerase can only synthesize new DNA in the 5' to 3' direction, the two new strands are built differently.
Feature | Leading Strand | Lagging Strand |
|---|---|---|
Template orientation relative to fork | 3' to 5' toward the fork | 5' to 3' toward the fork |
Synthesis pattern | Continuous | Discontinuous (short fragments) |
Primary eukaryotic polymerase | DNA polymerase epsilon (Pol ε) | DNA polymerase delta (Pol δ), extending primers laid by Pol α |
Primer requirement | One primer, at the origin | Repeated primers, one per fragment |
Because the leading strand's template runs 3' to 5' toward the fork, DNA polymerase can follow right behind helicase and synthesize continuously — in eukaryotes, this is carried out primarily by DNA polymerase epsilon (Pol ε).
The lagging strand's template runs the opposite direction, so it can't be synthesized continuously. Instead, it's built in short fragments called Okazaki fragments, synthesized away from the replication fork:
Primase periodically lays down new RNA primers closer to the replication fork.
DNA polymerase alpha (Pol α) adds a short stretch of DNA to each primer.
DNA polymerase delta (Pol δ) takes over to extend the fragment.
Each fragment is synthesized 5' to 3', but the fragments as a group are made moving away from the fork.
Okazaki Fragments and DNA Ligase
Once primers are removed and gaps filled with DNA, the nicks between adjacent Okazaki fragments still need to be sealed. That's the job of DNA ligase, which forms phosphodiester bonds between fragments to create one continuous strand.
Removing RNA Primers
Eventually, the RNA primers on the lagging strand must be removed and replaced with DNA. In prokaryotes, DNA polymerase I removes RNA primers using its 5'-to-3' exonuclease activity and fills the resulting gap.
In eukaryotes, primer removal mainly happens through Pol δ's strand-displacement activity: as Pol δ extends a fragment, it displaces the downstream RNA primer into a short flap, which is then cut by FEN1 (flap endonuclease 1). RNase H2 assists this process by trimming most of the RNA portion of the primer beforehand. Once the primer is fully removed, Pol δ fills the gap with DNA nucleotides.
Telomere Synthesis (Eukaryotes Only)
One replication step applies only to eukaryotic cells: synthesizing telomeres. Because DNA polymerase can't fully replicate the very ends of linear DNA, eukaryotic cells rely on telomerase — an enzyme that extends chromosome ends using its own built-in RNA template.
Prokaryotes don't need this step because their chromosomes are circular; circular DNA has no ends to lose.
Semiconservative Replication
The entire replication process — unwinding, priming, synthesis, and ligation — is carried out by a multi-protein structure called the replisome (or replication complex), operating at both replication forks and across multiple replication bubbles in eukaryotic genomes.
The result is two identical daughter DNA molecules, each made of one original (template) strand and one newly synthesized strand. This is what's meant by semiconservative replication.
DNA Damage, Oncogenes, and Tumor Suppressor Genes
DNA can be damaged in many ways — some damage happens during replication itself, while other damage arises from environmental exposures like UV radiation, chemicals, or reactive oxygen species produced during normal metabolism. Unrepaired damage can cause mutations, and some mutations lead to uncontrolled cell growth and, ultimately, cancer.
Two gene categories are central to this process:
Oncogenes are mutated versions of normal genes called proto-oncogenes, which normally help regulate cell growth and division. A mutation can turn a proto-oncogene into an oncogene, making it overactive and driving unchecked cell division.
Tumor suppressor genes — like p53 and Rb — act as safeguards, halting the cell cycle or triggering DNA repair when damage is detected. Because of this protective role, they're also called anti-oncogenes. If a tumor suppressor gene is mutated and loses function, the cell loses that protective mechanism.
In short: cancer can arise from a gain of function in a proto-oncogene (turning it into an oncogene) or a loss of function in a tumor suppressor gene.
DNA Repair Mechanisms
DNA repair mechanisms fall into two major categories: Replication Error Repair, which fixes mistakes made during or just after replication, and Damaged Base Repair, which corrects chemical or environmental damage that can occur at any point in the cell cycle.
Proofreading
Proofreading happens as DNA polymerase synthesizes the new strand. If it detects a mismatched or unstable base pair, it pauses, excises the incorrect nucleotide, and replaces it with the correct one — catching errors right at the source.
Mismatch Repair
Mismatch repair scans for base-pairing errors that escaped proofreading. It's temporally coupled to replication and occurs shortly afterward, largely during S phase, while the cell can still distinguish the newly synthesized strand from the template strand (via methylation patterns). Once identified, the error is removed and replaced.
The key players are MSH2 and MLH1 — the human/eukaryotic homologs of the bacterial mismatch-repair proteins MutS and MutL. MSH2 (a MutS homolog) helps recognize the mismatch, and MLH1 (a MutL homolog) helps coordinate the repair.
Nucleotide Excision Repair
Nucleotide excision repair fixes bulky lesions, such as thymine dimers caused by UV radiation. An excision endonuclease cuts on both sides of the damaged section, DNA polymerase fills in the gap, and DNA ligase seals it.
Base Excision Repair
Base excision repair handles smaller, non-bulky damage, such as a deaminated cytosine. A glycosylase removes the damaged base, leaving behind an empty sugar-phosphate backbone called an AP site. An AP endonuclease then cuts the backbone at that site, and DNA polymerase and ligase fill and seal the gap.
Pathway | Category | Type of Damage | Key Enzymes |
|---|---|---|---|
Proofreading | Replication error repair | Mismatched base, caught during synthesis | DNA polymerase (intrinsic exonuclease activity) |
Mismatch repair | Replication error repair | Mismatched base, caught after synthesis (S phase) | MSH2/MLH1 (MutS/MutL homologs) |
Nucleotide excision repair | Damaged base repair | Bulky lesions (e.g., thymine dimers) | Excision endonuclease, DNA polymerase, DNA ligase |
Base excision repair | Damaged base repair | Small, non-bulky damage (e.g., deaminated cytosine) | Glycosylase, AP endonuclease, DNA polymerase, DNA ligase |
Common MCAT Mistakes
Mixing up which strand is continuous vs. discontinuous. The leading strand is synthesized continuously because its template runs 3' to 5' toward the fork; the lagging strand is discontinuous (Okazaki fragments) because its template runs the opposite direction and DNA polymerase can only synthesize 5' to 3'.
Assuming any single mutation causes cancer. Cancer typically requires a gain of function in a proto-oncogene (creating an oncogene) or a loss of function in a tumor suppressor gene (like p53 or Rb) — the two mechanisms work in opposite directions but both remove a check on cell division.
Confusing proofreading with mismatch repair. Proofreading happens in real time as DNA polymerase synthesizes the strand, catching errors immediately. Mismatch repair happens afterward, largely during S phase, and relies on distinguishing the new strand from the template via methylation.
Mixing up nucleotide excision repair and base excision repair. Nucleotide excision repair targets bulky lesions like thymine dimers using an excision endonuclease; base excision repair targets small, non-bulky damage like a deaminated cytosine using a glycosylase and AP endonuclease — different enzymes for different damage sizes.
MCAT-Style Concept Check
Question: A cell has sustained two types of DNA damage: Region 1 contains a thymine dimer caused by UV exposure, and Region 2 contains a single deaminated cytosine. Which repair pathways would most directly correct each type of damage?
A) Region 1 by mismatch repair; Region 2 by proofreading
B) Region 1 by base excision repair; Region 2 by nucleotide excision repair
C) Region 1 by nucleotide excision repair; Region 2 by base excision repair
D) Region 1 by proofreading; Region 2 by mismatch repair
Answer: C
Explanation: Nucleotide excision repair corrects bulky lesions such as thymine dimers, using an excision endonuclease to cut out the damaged section before DNA polymerase and ligase fill and seal the gap — matching Region 1. Base excision repair corrects smaller, non-bulky damage such as a deaminated cytosine, using a glycosylase to remove the damaged base and an AP endonuclease to cut the backbone at the resulting AP site — matching Region 2. Option B reverses these pairings. Options A and D are wrong because proofreading and mismatch repair correct replication errors (mismatched bases), not environmental chemical damage like thymine dimers or deaminated cytosine.
FAQ
What's the difference between the leading and lagging strand?
The leading strand's template runs 3' to 5' toward the replication fork, so DNA polymerase synthesizes it continuously. The lagging strand's template runs the opposite direction, so it's synthesized discontinuously as short Okazaki fragments, which are later joined by DNA ligase.
Why is telomerase only needed in eukaryotic cells?
Telomerase extends the ends of linear chromosomes, which DNA polymerase can't fully replicate on its own. Prokaryotic chromosomes are circular and have no ends to lose, so they don't face this problem and don't need telomerase.
What's the difference between an oncogene and a tumor suppressor gene?
An oncogene is a mutated, overactive version of a proto-oncogene that drives unchecked cell division — a gain of function. A tumor suppressor gene, like p53 or Rb, normally halts the cell cycle or triggers repair when damage is detected; cancer risk rises when it loses function through mutation.
How is nucleotide excision repair different from base excision repair?
Nucleotide excision repair removes bulky lesions, such as UV-induced thymine dimers, using an excision endonuclease to cut out a section of the strand. Base excision repair removes smaller, non-bulky damage, such as a deaminated cytosine, using a glycosylase to remove just the damaged base.
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