D. Molecular Genetics · Molecular GeneticsGetting It Right: Proofreading and DNA Repair
D3.1 D3.4 D2.1 A2.2About 65 minutes
Learning goals- I can explain how proofreading by DNA polymerase and mismatch repair keep replication accurate.
- I can describe base excision repair, nucleotide excision repair and double-strand break repair, and what kind of damage each fixes.
- I can calculate how error rates add up over a genome.
- I can connect failures of DNA repair to human diseases such as xeroderma pigmentosum and some inherited cancers.
Words to know
proofreading
The ability of a DNA polymerase to detect a wrongly paired nucleotide it has just added, remove it with its 3′→5′ exonuclease activity, and try again.
exonuclease
An enzyme that removes nucleotides one at a time from the end of a DNA strand.
mismatch repair
A system that finds wrongly paired bases left after replication and replaces a section of the new strand.
base excision repair
Removal and replacement of a single damaged or incorrect base, such as uracil in DNA.
nucleotide excision repair
Removal of a short stretch of a strand containing a bulky lesion, such as a thymine dimer, followed by resynthesis.
thymine dimer
Two adjacent thymines on the same strand joined by covalent bonds after absorbing UV light; it distorts the helix.
double-strand break
A cut through both strands of DNA, caused for example by ionizing radiation.
homologous recombination
Accurate repair of a double-strand break using an identical copy (the sister chromatid) as a template.
A billion letters with almost no typos
Imagine copying the 3.1 billion letters of one set of your chromosomes by hand. Even an excellent typist makes about one mistake per thousand characters, which would be 3 million errors. Your cells copy all 6.4 billion base pairs in a few hours and, after every check, leave only a handful of uncorrected errors. On top of that, every cell's DNA is damaged thousands of times a day by heat, chemicals in the cell and the environment, and radiation.
The curriculum (D3.1) asks you to describe the repair mechanisms that correct these mistakes. They are a big part of why most of your cells work normally for decades, and when they fail, mutations pile up.
Layer 1 and 2: choosing correctly, then proofreading
DNA polymerase picks each incoming nucleotide by how well it fits its partner on the template. A correct pair has the right shape and hydrogen bonds; a wrong one fits poorly. Even so, about 1 in 100 000 nucleotides added is wrong.
Many DNA polymerases, including E. coli DNA polymerase III and I, can proofread. If the base just added is mismatched, the 3′ end of the new strand does not sit properly in the enzyme. The polymerase pauses, moves the end into a second active site with 3′→5′ exonuclease activity, clips off the wrong nucleotide, and then adds the correct one. Proofreading improves accuracy about 100-fold, to roughly 1 in 10 million.
Layer 3: mismatch repair
Some mismatches escape proofreading. Right after replication, mismatch repair proteins scan the new DNA for the bulge a mismatch makes.
The tricky part: if G is paired with T, which one is wrong? The repair system must fix the new strand, because the parent strand carries the original information. In E. coli, an enzyme adds methyl groups to adenines in the sequence GATC, but only some minutes after replication. So for a short time, the parent strand is methylated and the new strand is not. The repair proteins (named MutS, MutL and MutH) use this to identify and cut the new strand. An exonuclease removes a section of it, including the wrong base; DNA polymerase III fills in the gap and ligase seals it. Eukaryotes use similar proteins and different signals to tell the strands apart.
Mismatch repair improves accuracy by another 100 to 1000 times, to about 1 error per billion (10⁹) nucleotides.
Repairing damage (not just copying errors)
DNA is also damaged between replications. Different kinds of damage have different repair systems.
Base excision repair (BER) fixes a single damaged base. For example, cytosine can lose an amino group (deamination) and become uracil, which pairs with A instead of G. A DNA glycosylase enzyme recognizes the uracil (which does not belong in DNA) and removes just the base. Other enzymes cut out the empty sugar, a polymerase inserts the correct nucleotide, and ligase seals the backbone.
Nucleotide excision repair (NER) fixes bulky damage that distorts the helix. The classic example is the thymine dimer: when DNA absorbs UV light, two thymines next to each other on the same strand become covalently bonded. Enzymes cut the damaged strand on both sides of the lesion (in E. coli, about 12 or 13 nucleotides are removed; in humans, about 24 to 32), DNA polymerase fills the gap using the undamaged strand as a template, and ligase seals it. Bulky chemical damage, such as from compounds in tobacco smoke, is also repaired this way.
Direct reversal. Some organisms, including bacteria and plants, have an enzyme called photolyase that uses the energy of visible light to break the bonds of a thymine dimer directly. Placental mammals, including humans, do not have it and rely on NER.
Double-strand break repair. Ionizing radiation (X-rays, gamma rays) and some chemicals can cut both strands. This is the most dangerous damage, because there is no intact partner strand on the spot to copy from.
- Non-homologous end joining (NHEJ) simply glues the broken ends back together. It is fast but often adds or loses a few bases.
- Homologous recombination uses the identical sister chromatid (available after replication) as a template, so it is accurate. The proteins made by the genes BRCA1 and BRCA2 help with this pathway.
You will see in the gene-editing part of this unit that CRISPR tools work by making a double-strand break and letting these repair systems do the rest.
| Repair system | Problem it fixes | Key steps |
|---|
| Proofreading | wrong nucleotide just added during replication | polymerase's 3′→5′ exonuclease removes it at once |
| Mismatch repair | mismatched pairs left after replication | identify new strand, excise a section, resynthesize |
| Base excision repair | one damaged or wrong base (e.g., uracil) | glycosylase removes base; backbone cut; refill; seal |
| Nucleotide excision repair | bulky lesions (e.g., thymine dimers) | cut both sides of lesion; remove piece; refill; seal |
| Double-strand break repair | both strands broken | end joining (fast, error-prone) or homologous recombination (accurate) |
When repair fails
Because repair systems are made of proteins, a mutation in a repair gene makes every other gene more likely to mutate.
- Xeroderma pigmentosum (XP): people with this inherited condition have a faulty NER enzyme, cannot repair UV damage well, and must avoid sunlight to reduce a very high risk of skin cancer.
- Lynch syndrome: an inherited mutation in a mismatch-repair gene increases the risk of colorectal and some other cancers.
- Hereditary breast and ovarian cancer: inherited mutations in BRCA1 or BRCA2 impair homologous recombination.
Scientists behind it: the 2015 Nobel Prize in Chemistry was shared by Tomas Lindahl (base excision repair), Paul Modrich (mismatch repair) and Aziz Sancar (nucleotide excision repair) for showing how cells repair DNA.
Worked exampleExample 1: How many errors per division?
A human diploid cell copies 6.4 × 10⁹ base pairs. Using the round numbers in this lesson, estimate the number of errors (a) with base selection only, (b) after proofreading, (c) after mismatch repair.
- (a) 6.4 × 10⁹ × (1/10⁵) = 6.4 × 10⁴, about 64 000 errors.
- (b) 6.4 × 10⁹ × (1/10⁷) = 640 errors.
- (c) 6.4 × 10⁹ × (1/10⁹) = 6.4, about 6 errors per division.
- Each layer removes most of the errors left by the layer before.
About 64 000, then 640, then about 6 errors per cell division.
Worked exampleExample 2: Which strand gets fixed?
After replication in E. coli, a G-T mismatch is found. The strand with the G is methylated at nearby GATC sites; the strand with the T is not. Which base is replaced, and what is the final base pair?
- The methylated strand is the parent (original) strand, so its base, G, is assumed to be correct.
- The unmethylated strand is new, so the section containing T is removed.
- Polymerase fills the gap using the parent strand as the template, putting C opposite G.
T is replaced; the final pair is G-C.
Just for funDNA polymerase had a working "backspace" key (its 3′→5′ exonuclease) billions of years before anyone invented a keyboard.
Hands-on activityLayers of accuracy: a copying experiment
You need: a printed or handwritten list of 200 random letters using only A, T, G and C (a family member can write it, in groups of 10), a pencil, paper, a timer and a family member.
- Base selection: copy all 200 letters as fast as you can, in 2 minutes. No looking back.
- Proofreading: go back over your copy once, comparing each group of 10 with the original, and fix any errors you find. Count how many you fixed.
- Mismatch repair: your family member checks the corrected copy letter by letter and marks any remaining errors. Count them.
- Calculate the error rate at each stage: errors ÷ 200. For example, 8 errors after step 1 = 8/200 = 1 in 25.
- How many times better did each layer make the copy?
What to look for: each check catches most, but not all, of the errors left by the step before, just as proofreading and mismatch repair do. Real DNA polymerase, before any checking, makes about 1 error per 100 000 nucleotides. Compare that with the error rate of your first copy.
Your turn
1What enzyme activity allows DNA polymerase III to proofread?
Its 3′→5′ exonuclease activity, which removes a wrongly paired nucleotide from the 3′ end of the new strand.
2How does the mismatch repair system in E. coli tell the new strand from the parent?
The parent strand is methylated at GATC sites; the new strand is not yet methylated, so the unmethylated strand is the one repaired.
3Which repair system removes uracil from DNA?
Base excision repair (a DNA glycosylase removes the uracil base).
4What is a thymine dimer, and which repair system fixes it in humans?
Two adjacent thymines on the same strand covalently bonded after absorbing UV light. In humans it is fixed by nucleotide excision repair.
5Why is a double-strand break more dangerous than a single-strand break?
With a single-strand break the other strand is intact and serves as a template. With a double-strand break, both strands are cut, the chromosome can fall apart, and repair may be inaccurate (end joining) or need a sister chromatid.
6Explain why a mutation in a DNA-repair gene can increase the rate of mutations in many other genes.
The repair gene codes for a repair protein. If that protein does not work, errors and damage anywhere in the genome are not fixed, so mutations accumulate in all genes, including those that control cell division.
More practice: warm-up
1About how often does DNA polymerase add a wrong nucleotide before proofreading?
About 1 in 100 000 (10⁵).
2What kind of radiation causes thymine dimers?
3What enzyme seals the backbone after repair?
4Name a disease caused by faulty nucleotide excision repair.
Xeroderma pigmentosum (XP).
5Which double-strand-break repair pathway uses the sister chromatid?
Homologous recombination.
More practice: on your own
1The E. coli genome has 4.6 × 10⁶ base pairs. With an overall error rate of 1 in 10⁹, about how many errors occur per replication? What does this mean for a population of bacteria?
4.6 × 10⁶ ÷ 10⁹ ≈ 0.005 errors per replication, about 1 error per 200 cell divisions. In a large population (billions of cells), many new mutations still appear every generation, which supplies variation for evolution (and for antibiotic resistance).
2Compare base excision repair and nucleotide excision repair.
Both remove damage and refill the gap using the other strand as a template, then seal with ligase. BER removes a single damaged base (via a glycosylase); NER removes a stretch of nucleotides (about 12 to 32) around a bulky helix-distorting lesion.
3Why must the methyl groups be added to the new strand only after a delay for mismatch repair to work?
If the new strand were methylated immediately, both strands would look the same and the repair system could not tell which base was the original. The delay gives a short window in which only the parent strand is methylated.
4Explain why non-homologous end joining is described as error-prone.
It joins broken ends directly without a template. The ends are often trimmed or have a few bases added before joining, so small insertions or deletions can be introduced at the repair site.
5Why do people with xeroderma pigmentosum have a high risk of skin cancer but not a high risk of most internal cancers?
Their defect is in repairing UV damage (thymine dimers). UV light reaches the skin and eyes but not internal organs, so the extra mutations happen mainly in sun-exposed cells.
Challenge
1A mutant E. coli strain lacks the enzyme that methylates GATC sites. Predict the effect on mismatch repair and on its mutation rate.
Without methylation, neither strand is marked, so the repair system cannot tell new from parent. It will often 'repair' the wrong strand, so many mismatches become permanent mutations (about half the time the parent base is replaced). The mutation rate would increase.
2Some cancer drugs (PARP inhibitors) block a single-strand repair pathway. They are especially effective in tumours with BRCA1 or BRCA2 mutations. Using what you know about repair pathways, suggest why.
If single-strand breaks are not repaired, they turn into double-strand breaks when the DNA is replicated. Normal cells repair these accurately by homologous recombination, but BRCA-mutant tumour cells cannot. The breaks pile up and the cancer cells die, while normal cells (with working BRCA) survive. This is called synthetic lethality.
3A biology student says, "Since mutations are harmful, the best organisms would have a mutation rate of zero." Evaluate this statement.
Partly true for an individual: most mutations that change proteins are harmful or neutral, and high mutation rates cause disease such as cancer. But for a species, some mutation is needed to produce the genetic variation that natural selection acts on. Also, perfect accuracy would cost time and energy. Real organisms balance accuracy against speed and cost; zero is neither achievable nor clearly best.
Quiz
1. Which activity allows DNA polymerase to remove a nucleotide it has just added incorrectly?
2. In E. coli, mismatch repair identifies the new strand because it:
3. Which type of damage is fixed by nucleotide excision repair?
4. Using an overall error rate of 1 in 10⁹, about how many errors remain after copying 6.4 × 10⁹ base pairs?
5. Which double-strand-break repair pathway is accurate because it copies the sister chromatid?
6. People with xeroderma pigmentosum are very sensitive to sunlight because they:
Big idea (D3.1, with D3.4): cells keep DNA accurate with layers of protection: careful base selection, proofreading, mismatch repair, and several systems that repair damage. When these systems fail, mutations accumulate, which can lead to cancer.
How to help: Ask your teen to match each kind of damage to its repair system (wrong base during copying → proofreading or mismatch repair; uracil → base excision; UV dimer → nucleotide excision; both strands cut → end joining or recombination).
Common difficulties:
- Confusing "proofreading" (during replication, by the polymerase) with "mismatch repair" (after replication, by separate proteins).
- Not knowing how the cell decides which strand is wrong (methylation in E. coli).
- Treating DNA damage and mutation as the same thing. Damage that is repaired correctly never becomes a mutation.
Some families have inherited conditions such as Lynch syndrome or BRCA mutations. If this topic is personal, genetic counsellors can answer family-specific questions.