DNA Polymerase is the enzyme responsible for copying the human genome, a process known as DNA replication. This enzyme synthesizes a new DNA strand by matching incoming nucleotides—the building blocks of DNA—to the template strand. Its primary function is to extend the growing DNA chain in the 5′ to 3′ direction, ensuring that adenine pairs with thymine and guanine pairs with cytosine. The genetic blueprint must be passed on flawlessly, making the fidelity of this copying process paramount for the survival and proper function of the organism.
The Requirement for High-Fidelity Replication
DNA Polymerase, relying only on the basic chemical fit between bases, would make a mistake about once every 10,000 to 100,000 nucleotides added. The enzyme’s initial base selection mechanism is not perfectly accurate, occasionally allowing a mismatched nucleotide to be incorporated. While this error rate might seem low, it is far too high when considering the sheer size of a typical genome. The human genome contains approximately three billion base pairs.
Without further correction, every round of cell division would introduce tens of thousands of errors into the genetic code. This influx of mistakes would quickly compromise the integrity of the genome and the function of resulting proteins. Therefore, an immediate, intrinsic correction mechanism is required to reduce this baseline error rate to a manageable level. A self-checking capacity evolved within the enzyme itself to ensure genetic information remains stable across generations.
The 3′ to 5′ Exonuclease Proofreading Activity
DNA Polymerase possesses a proofreading function that enhances replication accuracy. It corrects the enzyme’s own errors immediately after they occur. The core of this self-correction mechanism is a separate domain within the polymerase called the 3′ to 5′ exonuclease.
When a mismatched nucleotide is incorporated, the incorrect pairing causes a structural distortion that the polymerase detects. Sensing this mismatch, the enzyme temporarily halts its forward synthesis and shifts the newly synthesized strand from the polymerization site to the exonuclease domain. The exonuclease cleaves the phosphodiester bond, removing the incorrectly added nucleotide from the 3′ end of the strand.
Error removal occurs in the 3′ to 5′ direction, opposite to DNA synthesis (5′ to 3′). Once excised, the strand moves back to the polymerization domain, where the polymerase inserts the correct nucleotide and resumes synthesis. This proofreading process reduces the overall error rate of replication by a factor of 100 to 1000-fold, resulting in a low final error rate of about one mistake per ten million to one hundred million base pairs.
Genetic Stability and the Impact of Proofreading Failure
The proofreading mechanism is a major factor in maintaining genetic stability. Errors bypassing the initial base selection and 3′ to 5′ exonuclease proofreading become permanent mutations. These uncorrected changes can alter the blueprint for proteins, potentially leading to cellular dysfunction or uncontrolled growth.
A failure in the proofreading domain of DNA polymerases has severe biological consequences. For example, mutations in the exonuclease domain of the human DNA Polymerase epsilon (POLE) gene lead to a hyper-mutated genotype. Cells with this defect accumulate errors at an extraordinarily high rate, sometimes exceeding 100 mutations per million bases.
This increase in the mutation rate is associated with a higher risk of developing certain diseases, particularly ultra-mutated human cancers such as colorectal and endometrial tumors. The existence of these cancer-associated mutations underscores the importance of the proofreading function as a primary guardian of the cell’s genetic material.