In this article we will discuss about the fidelity of the replication of DNA.
The DNA being the carrier of genetic information it is essential that this information remains correct. But the DNA can be damaged by the action of chemical or physical agents. The cell therefore possesses repair systems which can maintain the integrity of genetic information.
It is also of utmost importance that during the replication, the newly synthesized DNA be an exact copy, correct to one nucleotide, of the template DNA. Replication must therefore be a faithful process i.e. it should make no errors.
If such errors did exist, they could be detected by the repair systems but the latter would not be able to distinguish between the strand carrying the correct information and the one carrying the erroneous information. It is therefore necessary to have a corrector activity during the synthesis.
Corrector Activity of DNA Polymerases:
Given that the genome of E.coli comprises about 4 x 106 base pairs, the admissible rate of error for a totally correct replication is one erroneous base for every 106, or even 107, base pairs. The mere complementarity of bases will not ensure such a low rate of error because the bases can tautomerize.
Their enol form (unusual in physiological conditions) can pair with a normally non complementary base: e.g., a cytosine with adenine. It is believed that the frequency of these enolic forms is 10-4 to 10-5 base pairs. It may be noted therefore that without a correction mechanism, the fidelity of replication would be 10-4 or 10-5 and not 10-7.
The correction mechanism was extensively studied in E.coli. It is based on the fact that the DNA polymerases need both a primer and a template. In other words, the polymerase will not be able to bind a new nucleotide to the 3′ OH of a nucleotide not paired to the template DNA. If a base in its enol form has been incorporated, its return to the keto form results in a non-pairing.
Therefore, the DNA polymerase can no longer catalyze the formation of a phosphodiester bond with the following nucleotide. Its 3′ → 5′ exonuclease activity eliminates the ill-paired nucleotide, thus liberating a 3’OH utilizable by the DNA polymerase. A mutation of sub-unit e, carrying the 3′ → 5′ exonuclease activity, generates a DNA polymerase III which makes many more errors.
For eucaryotes, the problem of fidelity of replication becomes even more crucial, because the genome of a mammal for example, contains about 3 X 109 base pairs. The fidelity of replication must therefore be equal to, or greater than 10-9. The existence of an exonuclease activity 3′ → 5′ associated with DNA polymerase d and perhaps with DNA polymerase α explains this fidelity to a large extent.
It is generally admitted that the presence of auxiliary proteins and the very nature of the eucaryote chromosome permit this very low rate of error.
In this sense, it has been observed that the error rate of DNA polymerases increases when the enzymes are in experimental conditions in which they synthesize the DNA in a relatively continuous manner (they are then said to be processive). This suggests that in their active conformation these enzymes are very faithful.
Fidelity and Mechanism of Replication:
As just seen, the fact that a DNA polymerase has an absolute requirement for a primer 3’OH paired with the strand of the template DNA to synthesize a new DNA chain, allows the corrector activity to fully play its role.
It may also be observed that if one of the two strands of the DNA were copied by a DNA polymerase acting in the direction 3′ → 5′ that copy would be less faithful.
As a matter of fact, in this case, the growing chain would carry the triphosphate and the nucleotide to be added would carry the 3’OH. After the hydrolysis of an ill-paired nucleotide, there would be no more triphosphate available on the growing chain to permit the addition of a new nucleotide.
If the first nucleotide incorporated were a deoxyribonucleoside triphosphate, it could never be corrected by the exonuclease activity. At the end of the synthesis, the ribonucleotide primer will be systematically recognized and eliminated; as a result, the fact that the RNA polymerases are not very faithful (10-4 to 10-5) loses its importance.
One can realize that the very complexity of the replication mechanism (necessity of a primer, synthesis only in the 5′ → 3′ direction, ribonucleic nature of the primer) provides a corrector activity at the precise moment of replication, and sufficient fidelity for maintaining the exact genetic information.