6.5: Proofreading

Synthesis of new DNA molecules starts when DNA polymerase links nucleotides together in a sequence that is complementary to the template DNA strand. DNA polymerase has a higher affinity for the correct base to ensure fidelity in DNA replication. The DNA polymerase furthermore proofreads during replication, using an exonuclease domain that cuts off incorrect nucleotides from the nascent DNA strand.

Errors during Replication Are Corrected by the DNA Polymerase Enzyme

Genomic DNA is synthesized in the 5’ to 3’ direction. Each cell contains a number of DNA polymerases that play different roles in synthesizing and correcting mistakes in DNA; DNA polymerase delta and epsilon possess proofreading ability when replicating nuclear DNA. These polymerases “read” each base after it is added to the new strand. If the newly-added base is incorrect, the polymerase reverses direction (moving from 3’ to 5’) and uses an exonucleolytic domain to cut off the incorrect base. Subsequently, it is replaced with the correct base.

Mutations in the Exonuclease Domain of DNA Polymerase Are Linked to Cancers

Proofreading is important for preventing mutations from occurring in newly-synthesized DNA, but what happens when the proofreading mechanism fails? When a mutation alters the exonuclease domain of DNA polymerase, it loses the ability to remove incorrect nucleotides. In consequence, mutations can accumulate rapidly throughout the genome. This type of mutation has been linked to various types of cancer.

Low-Fidelity DNA Polymerase Can Generate Mutated DNA Sequences

Modified DNA polymerases are used in laboratory science for polymerase chain reaction (PCR), an in vitro technique for making many copies of specific fragments of DNA. While high-fidelity polymerases are used when it is important that the end product is perfect, some techniques, such as error-prone PCR, seek to generate mutations in a stretch of DNA on purpose. These techniques use polymerases that have compromised proofreading ability.

During DNA replication, nucleotides are normally added to the new strand in a sequence complimentary to the template. With adenosine binding to thymine, and cytosine binding to guanine.

However, nucleotides can be incorrectly paired. For instance, adenosine with cytosine. These errors can be prevented or fixed during replication, by a series of proofreading steps carried out by DNA polymerase, the enzyme that synthesizes DNA.

First, DNA polymerase has a higher affinity for correctly paired nucleotides, which decreases the likelihood of incorrect pairings.

Second, as nucleotides start to be paired to the template, DNA polymerase undergoes a conformational change, that makes incorrectly paired nucleotides more likely to disassociate allowing the correct nucleotides to be added.

Third, if an incorrect nucleotide manages to be added to a growing DNA chain it will not be paired correctly to the template due to structural issues. This incorrect pairing on the three prime end of the growing chain causes DNA synthesis to pause.

The three prime end will then move to a specific exonuclease site on DNA polymerase that removes nucleotides in the three prime to five prime direction. In this is exonucleolytic proofreading step, the mispaired end is removed and then replaced by the correct nucleotides.