The Fidelity of DNA Replication

By Dillon Lim - Medicine Student @ Brasenose College, Oxford

 

A DNA replication mechanism with a high-fidelity is essential to protect the genome from deleterious, i.e. harmful mutations. Mutations can affect protein-coding sequences, which directly affect the Order of amino acids put together in protein synthesis, but also regulatory sequences, and these errors can be equally if not more harmful. The error rate when we carry out synthesis is thankfully acceptably low – one in every 109 nucleotides. There are three main ways in which we do this – complimentary base pairing, proofreading, and mismatch repair.


DNA tends to form “Watson-Crick” pairings – adenine with thymine and cytosine with guanine. The pairings are stabilised by hydrogen bonds which secure the conformation of the two bases on opposite strands of the molecule. It is important to note however that other combinations are possible – not only are there other nucleobases (uracil and hypoxanthine are two examples) but also different conformations in which they can interact; hydrogen bonds are still formed between the non-Watson-Crick pairing.


So how do we select for the right pairing? It appears that DNA polymerases have a means of selecting bases geometrically. It is possible that having the right nucleotide bind to the polymerase’s active site – in the correct orientation and distance away from the adjacent nucleotide on the template strand – causes the conformational change that allows for the best catalysis of polymerisation. Experimentally, we see that several chemical analogues of bases can be incorporated into a DNA molecule by polymerases, even if they have poor hydrogen bond forming activity. We also know that the most common mismatches in DNA replication are indeed the ones with the closest geometry to the conventional “correct” pair.


You may be familiar from your chemistry that for a reaction to be spontaneous, it must have a negative free energy. The problem is that while the formation of a Watson-Crick pair is enthalpically favoured, the difference in free energy for the reaction of different isosteric bases with a given base is not significant because of a large drop in entropy, and because water as a polar molecule tends to weaken the strength of hydrogen bonding. Polymerase active sites therefore clamp down on the DNA strand – this not only helps to extrude ≈40% of water (strengthening hydrogen bonding) but also by restricting the movement of molecules within the active site reduces the contribution of entropy to the free energy equation.


Several polymerases have a 3’ -> 5’ exonuclease (i.e. cutting out bits of DNA) activity, on top of their ‘main’ 5’ -> 3’ polymerase function. This acts as a proofreading mechanism directly after a nucleotide is added. There is a kind of competition between the two active sites to bind to the DNA molecule – it is believed that the binding of an incorrect nucleotide reduces the affinity of the molecule for the polymerisation site and increases the affinity of the molecule for the exonuclease site, allowing it to cut out the incorrect base.


Mismatch repair is a fidelity mechanism that actually takes place after replication of DNA is complete. wide variety of proteins are involved, with different complexes for mispairs and indels.


Further reading:

  1. The High Fidelity of DNA Replication. https://www.scientificamerican.com/article/the-high-fidelity-of-dna-duplicatio/

  2. Fidelity of DNA replication - a matter of proofreading. https://link.springer.com/article/10.1007/s00294-018-0820-1 (Advanced)

  3. DNA Replication - A Matter of Fidelity. https://www.sciencedirect.com/science/article/pii/S109727651630140X (Advanced)