Proofreading and Repair

Proofreading and Repair

DNA replication takes place only once each generation in each cell, unlike other processes, such as RNA and protein synthesis, which occur many times. It is essential that the fidelity of the replication process be as high as possible to prevent mutations, which are errors in replication. Mutations are frequently harmful, even lethal, to organisms. Nature has devised several ways to ensure that the base sequence of DNA is copied faithfully.

Errors in replication occur spontaneously only once in every 10⁹ to 10¹⁰ base pairs. Proofreading refers to the removal of incorrect nucleotides immediately after they are added to the growing DNA during the replication process. DNA polymerase I has three active sites, as demonstrated by Hans Klenow. Pol I can be cleaved into two major fragments. One of them (the Klenow fragment) contains the polymerase activity and the proofreading activity. The other con-tains the 5' - > 3' repair activity. Figure 10.10 shows the proofreading activity of Pol I. Errors in hydrogen bonding lead to the incorporation of an incorrect nucleotide into a growing DNA chain once in every 10⁴ to 10⁵ base pairs. DNA polymerase I uses its 3' exonuclease activity to remove the incorrect nucleotide. Replication resumes when the correct nucleotide is added, also by DNA poly-merase I. Although the specificity of hydrogen-bonded base pairing accounts for one error in every 10⁴ to 10⁵ base pairs, the proofreading function of DNA polymerase improves the fidelity of replication to one error in every 10⁹ to 10¹⁰ base pairs.

How does proofreading improve replication fidelity?

During replication, a cut-and-patch process catalyzed by polymerase I takes place. The cutting is the removal of the RNA primer by the 5' exonuclease function of the polymerase, and the patching is the incorporation of the required deoxynucleotides by the polymerase function of the same enzyme. Note that this part of the process takes place after polymerase III has produced the new polynucleotide chain. Existing DNA can also be repaired by polymerase I, using the cut-and-patch method, if one or more bases have been damaged by an external agent, or if a mismatch was missed by the proofreading activity. DNA polymerase I is able to use its 5' - > 3' exonuclease activity to remove RNA primers or DNA mistakes as it moves along the DNA. It then fills in behind it with its polymerase activity. This process is called nick translation (Figure 10.11). In addition to experiencing those spontaneous mutations caused by misreading the genetic code, organisms are frequently exposed to mutagens, agents that produce mutations. 

Common mutagens include ultraviolet light, ionizing radiation (radioactivity), and various chemical agents, all of which lead to changes in DNA over and above those produced by spontaneous mutation. The most common effect of ultraviolet light is the creation of pyrimidine dimers (Figure 10.12). The π electrons from two carbons on each of two pyrimidines form a cyclobutyl ring, which distorts the normal shape of the DNA and interferes with replication and transcription. Chemical damage, which is often caused by free radicals (Figure 10.13), can lead to a break in the phosphodiester backbone of the DNA strand. This is one of the primary reasons that antioxidants are so popular as dietary supplements these days.

When damage has managed to escape the normal exonuclease activities of DNA polymerases I and III, prokaryotes have a variety of other repair mecha-nisms at their disposal. In mismatch repair, enzymes recognize that two bases are incorrectly paired. The area with the mismatch is removed, and DNA polymerases replicate the area again. If there is a mismatch, the challenge for the repair system is to know which of the two strands is the correct one. This is possible only because prokaryotes alter their DNA at certain locations by modifying bases with added methyl groups. This methylation occurs shortly after replication. Thus, immediately after replication, there is a window of opportunity for the mismatch-repair system. Figure 10.14 shows how this works. 

Assume that a bacterial species methylates adenines that are part of a unique sequence. Originally, both parental strands are methylated. When the DNA is replicated, a mistake is made, and a T is placed opposite a G (Figure 10.14a). Because the parental strand contained methylated adenines, the enzymes can distinguish the parental strand from the newly synthesized daughter strand without the modified bases. Thus, the T is the mistake and not the G. Several proteins and enzymes are then involved in the repair process. MutH, MutS, and MutL form a loop between the mistake and a methylation site.DNA helicase II helps unwind the DNA. Exonuclease I removes the section of DNA containing the mistake (Figure 10.14b). Single-stranded binding proteins protect the template (blue) strand from degradation. DNA polymerase III then fills in the missing piece (Figure 10.14c).

Another repair system is called base-excision repair (Figure 10.15). A base that has been damaged by oxidation or chemical modification is removed by DNA glycosylase, leaving an AP site, so called because it is apurinic or apy-rimidinic (without purine or pyrimidine). An AP endonuclease then removes the sugar and phosphate from the nucleotide. An excision exonuclease then removes several more bases. Finally, DNA polymerase I fills in the gap, and DNA ligase seals the phosphodiester backbone.

Nucleotide-excision repair is common for DNA lesions caused by ultravioletor chemical means, which often lead to deformed DNA structures. Figure 10.16 demonstrates how a large section of DNA containing the lesion is removed by ABC excinuclease. DNA polymerase I and DNA ligase then work to fill in the gap.This type of repair is also the most common repair for ultraviolet damage in mammals. Defects in DNA repair mechanisms can have drastic consequences. One of the most remarkable examples is the disease xeroderma pigmentosum. Affected individuals develop numerous skin cancers at an early age because they do not have the repair system to correct damage caused by ultraviolet light. 

The endonuclease that nicks the damaged portion of the DNA is prob-ably the missing enzyme. The repair enzyme that recognizes the lesion has been named XPA protein after the disease. The cancerous lesions eventually spread throughout the body, causing death.

Summary

Bases would be paired incorrectly during DNA synthesis about once for every 10⁴ to 10⁵ base pairs unless there were a mechanism to increase fidelity.

Because of proofreading and repair, the number of incorrect bases is reduced to one in 10⁹ to 10¹⁰.

Proofreading is the process by which DNA polymerase I removes incor-rectly paired bases immediately after they are added to the growing chain.