Questions: DNA Proofreading and Mismatch Repair

Question 1

A mutation eliminates the 3'→5' exonuclease activity of DNA polymerase III but leaves its polymerization activity intact. What would you predict?

• A. Mutation rate would be unchanged — the polymerase's selectivity for correct nucleotides during incorporation is already sufficient for high fidelity
• B. Mutation rate would increase approximately 100-fold — proofreading provides the second layer of fidelity beyond nucleotide selection, and removing it eliminates correction of errors that escaped initial selectivity
• C. Mutation rate would increase approximately 10⁹-fold, wiping out genome stability entirely
• D. Mutation rate would decrease — the polymerase would advance more smoothly without pausing to backtrack

Answer: (B)

Nucleotide selection by the polymerase achieves approximately 1 error per 10⁵ bases. Proofreading (3'→5' exonuclease) then catches and removes most of the remaining mismatches, improving fidelity ~100-fold to ~1 per 10⁷. Removing proofreading restores the ~10⁵ error rate. Option A is wrong because selectivity alone is insufficient — mismatches do occur during initial incorporation and require proofreading to catch them. Option C overstates the effect: even without proofreading, mismatch repair would still provide another ~100-1000x improvement.

Question 2

In E. coli, a mutation eliminates MutH protein. MutS and MutL are still functional. What step of mismatch repair would specifically fail?

• A. Mismatch recognition would fail — MutH is required for MutS to bind mismatched bases
• B. Strand discrimination would fail — MutH nicks the newly synthesized (unmethylated) strand at GATC sequences; without it, the repair system cannot identify which strand contains the error and risks repairing the wrong strand
• C. Exonuclease removal of mismatched nucleotides would fail — MutH directly degrades the incorrect bases
• D. DNA re-synthesis after excision would fail — MutH activates the repair polymerase

Answer: (B)

MutS detects the mismatch; MutL coordinates the response. MutH performs the critical and unique step of strand discrimination: it nicks the hemimethylated GATC site on the transiently unmethylated new strand, identifying it as the error-containing strand for repair. Without MutH, the system detects the mismatch but cannot determine which of the two strands is wrong. Repairing the template strand would replace the correct base to match the error — permanently fixing the mutation rather than correcting it.

Question 3

The strand discrimination mechanism in E. coli mismatch repair exploits the transient hemimethylated state of newly replicated DNA: the template strand is already methylated at GATC sequences, while the newly synthesized strand is not yet methylated.

• T. True
• F. False

Answer: True

This is the elegant molecular solution to the 'which strand is wrong?' problem. Dam methylase adds methyl groups to adenine in GATC sequences, but there is a brief window after replication when only the template strand carries these marks — the new strand hasn't been methylated yet. MutH recognizes this hemimethylated state and nicks the unmethylated (new) strand, directing repair to the error-containing strand. This timing-based mechanism is critical: without it, the repair system could not reliably distinguish template from new strand.

Question 4

DNA polymerase proofreading and mismatch repair detect errors by the same molecular mechanism — recognizing incorrect Watson-Crick base pairs by their chemical properties.

• T. True
• F. False

Answer: False

These mechanisms are distinct. Polymerase proofreading detects geometric distortion in the polymerase's active site during synthesis — a mismatched nucleotide physically doesn't fit the active site's shape, causing the polymerase to stall and backtrack. Mismatch repair (via MutS) detects distortions in the backbone geometry of the already-synthesized double helix as MutS slides along DNA from outside the polymerase. One is an active-site structural check during synthesis; the other is a post-synthesis scanning mechanism. They are mechanistically independent, which is why removing one leaves the other fully functional.

Question 5

Why is the ability to distinguish the newly synthesized strand from the template strand essential for mismatch repair, and how does E. coli solve this problem?

Model answer: When MutS detects a mismatch, both strands contain a base at that position — but only one is wrong (the newly synthesized strand). If the repair machinery repaired the template strand instead, it would change the correct base to match the erroneous one, permanently converting the mismatch into a mutation. E. coli solves this using methylation: Dam methylase methylates adenine in GATC sequences on both strands, but newly replicated DNA is transiently hemimethylated — template strand methylated, new strand not yet. MutH recognizes this state and nicks the unmethylated strand, directing all repair activity to the strand most likely to contain the error.

Strand discrimination is the conceptually most subtle part of mismatch repair. Detecting a mismatch is the easy part — any distortion in the helix will do. Knowing which of the two bases is wrong requires an external signal, because the mismatched bases themselves don't tell you which is 'right.' The methylation-based system is a temporal trick: it exploits the gap between replication (which produces the new strand) and methylation (which eventually marks it) as a window during which the new strand is identifiable. Lynch syndrome, caused by defects in human mismatch repair genes, illustrates what happens when this system fails at scale.