DNA polymerase is the enzyme responsible for synthesizing new strands of DNA during replication. To ensure the genetic blueprint is accurately copied, the enzyme possesses a built-in corrective mechanism known as proofreading. This process operates immediately during replication to dramatically lower the error rate and ensure high fidelity.
The \(3′ \to 5′\) Exonuclease Activity
The ability of a DNA polymerase to proofread is due to a separate enzymatic function called \(3′ \to 5′\) exonuclease activity. The polymerase itself has a \(5′ \to 3′\) activity, which is the direction it synthesizes the new DNA strand. When an incorrect nucleotide is mistakenly incorporated into the growing chain, the enzyme detects the resulting structural distortion or mismatch at the active site.
Upon detection of a mismatched base pair, the polymerase stalls. The newly synthesized DNA strand’s \(3′\) end, which contains the error, then shifts into a separate active site on the enzyme known as the exonuclease domain. This domain acts as a molecular “backspace” function, moving backward along the DNA strand to remove nucleotides in the \(3′\) to \(5′\) direction. It specifically excises the incorrect, mismatched nucleotide from the end of the growing strand.
Once the wrong base is removed, the \(3′\) end of the DNA strand returns to the original polymerase active site. The enzyme is then able to correctly incorporate the proper base, and DNA synthesis resumes in the \(5′ \to 3′\) direction. This entire proofreading process reduces the error rate of DNA replication by a factor of 100 to 1,000-fold, ensuring genetic stability.
Primary Proofreading Enzymes in Bacteria
In prokaryotes, multiple DNA polymerase enzymes are involved in replication, and several possess proofreading capability. The main enzyme responsible for the bulk of chromosomal replication is DNA Polymerase III (Pol III). Pol III is a complex enzyme that is highly processive.
The proofreading function of DNA Polymerase III is carried out by a specific subunit within the enzyme complex, known as the epsilon (\(\epsilon\)) subunit, which contains the \(3′ \to 5′\) exonuclease activity. This subunit works in close cooperation with the alpha (\(\alpha\)) subunit, which is the polymerase domain responsible for synthesis. This coordinated effort ensures that errors are corrected in real-time as the bacterial chromosome is duplicated.
DNA Polymerase I (Pol I) is another bacterial enzyme that possesses the \(3′ \to 5′\) exonuclease proofreading activity. Pol I plays a crucial role in removing the RNA primers that initiate replication and filling in the resulting gaps on the lagging strand. While Pol III performs the main replicative synthesis, Pol I’s proofreading function contributes to the overall fidelity during this cleanup and repair process.
A third enzyme, DNA Polymerase II (Pol II), also has intrinsic \(3′ \to 5′\) exonuclease activity. Pol II is primarily a repair enzyme that can serve as a backup to Pol III if the main replicative polymerase is stalled or damaged.
Error Correction by Eukaryotic Polymerases
The process of DNA replication in eukaryotes is more complex and involves a greater number of polymerase enzymes. However, the fundamental mechanism of \(3′ \to 5′\) exonuclease proofreading remains the same for the high-fidelity replicative polymerases. The two primary enzymes responsible for synthesizing the vast majority of the eukaryotic genome are DNA Polymerase delta (\(\delta\)) and DNA Polymerase epsilon (\(\epsilon\)).
DNA Polymerase \(\delta\) and DNA Polymerase \(\epsilon\) are classified as high-fidelity enzymes, and both contain the necessary \(3′ \to 5′\) exonuclease domain to perform proofreading during elongation. Pol \(\epsilon\) is thought to primarily synthesize the leading strand, while Pol \(\delta\) is the main enzyme for lagging strand synthesis, but both require their proofreading domains to ensure the integrity of the new DNA.
It is important to note that not all eukaryotic polymerases possess this proofreading capability. For instance, DNA Polymerase alpha (\(\alpha\)) is involved in initiating replication by synthesizing a short RNA-DNA primer, but it lacks the \(3′ \to 5′\) exonuclease activity. Because Pol \(\alpha\) only synthesizes a very short segment of DNA before handing off to Pol \(\delta\) or Pol \(\epsilon\), its lower fidelity does not significantly compromise the overall accuracy of replication.