Study Notes on Fidelity of Replication | Cell Biology

Study notes on Fidelity of Replication!

Fidelity of replication means the faithful repli­cation of DNA and the production of accurate daughter DNA using the parental DNA as a template. The accuracy of DNA replication is critical to cell reproduction. To produce a sequence of bases in the DNA of a daughter cell which is identical to that in the parent, the process of DNA replication would need to be completely faithful.

This is obviously not the case as mutants do arise spontaneously. Mutation causes errors during replication. This error frequency is much lower than would be predicted simply on the basis of complemen­tary base pairing.

In particular, the stan­dard configurations of nucleic acid bases are in equilibrium with rare alternative conformations (tautomeric forms) that hydrogen-bond with the wrong partner (e.g., G with T) with a frequency of about one incorrect base per 10⁴.

Fidelity of replication may depend on a number of factors and also on a number of steps.

The much higher degree of fidelity actually achieved results largely from the activities of DNA polymerase. The mechanism by which DNA polymerase increases the fidelity of replication is by helping to select the correct base for insertion into newly synthesised DNA.

DNA polymerases are able to discriminate the mismatched base from the population of correct bases and are able to reject the incorrect base. Such discrimination power may result from template-induced changes in enzyme con­formation dictating the selection of the correct substrate or increased binding of the enzyme to the template in the presence of the comple­mentary nucleotide.

This selectivity appears to increase the accurracy of replication about a hundredfold and reduces the expected error frequency from 10^-4 to approximately 10~⁶.

The other major mechanism responsible for the fidelity of DNA replication is the proof­reading activity of DNA polymerases. It has been previously stated that DNA poly­merase I of E.coli has 3′ to 5′ as well as 5′ 3′ exonuclease activity.

The 5′ to 3′ exonuclease operates in the direction of DNA synthesis and helps remove RNA primers from Okazaki fragments. The 3′ and 5′ exonuclease acts in the reverse direction of DNA synthesis and par­ticipates in proof-reading of newly synthesised DNA.

Proof-reading is effective because DNA polymerase requires a primer and is not able to start synthesis de novo. Primers that are hydro­gen bonded to the template are preferentially used when an incorrect base is incorporated, it is likely to be removed by the 3′ -» 5′ ex­onuclease activity.

Such 3′ 5′ exonuclease activities are also associated with prokaryotic polymerase III and eukaryotic polymerases <5 and £. The 3′ ->■ 5′ exonucleases of these polymerases precisely cut the mismatched bases that have been incorporated at the end of a growing DNA chain, thereby increasing the accuracy of replication.

The importance of proof-reading may explain the fact that DNA polymerases need primers and catalyse the growth of DNA strand only in the 3′ 5′ direction.

When DNA is synthesised in the 5′ -¥ 3′ direc­tion, the energy needed for polymerisation is obtained from hydrolysis of the 5′ triphosphate group of free dNTP as it is added to the 3′ hy- droxyl group of the growing chain.

If DNA was to elongated in the 3′ 5′ direction, the energy of polymerisation would instead have to be come from hydrolysis of 5′ triphosphate group of the terminal nucleotide already incorporated into DNA. This would eliminate the possibility of proof-reading.

This is because removal of a mismatched terminal nucleotide would also eliminate the 5′ triphosphate group required as an energy source for further chain extension. Therefore, albeit the ability of DNA polymerase to extend a primer only in the 5′ 3′ direction appears to make replication, it is necessary for ensuring accurate duplication of DNA.

Therefore, all such error avoidance and post- replicative error correction mechanisms can in­crease the fidelity of replication.