Our genetic material, DNA, is constantly exposed to threats that can cause damage or errors. Maintaining the stability and integrity of this molecule is important for all living organisms. Cells have evolved sophisticated mechanisms to safeguard their DNA. Among these, 3′-5′ exonuclease activity stands out as a crucial guardian, preserving the accuracy of our genetic blueprint.
Decoding 3-5 Exonuclease Activity
An exonuclease is an enzyme that removes nucleotides. The “3′-5′” designation refers to the directionality of this removal, starting at the 3′ end and proceeding towards the 5′ end. This directionality is directly related to how DNA is synthesized. DNA polymerases, which build new DNA strands, add nucleotides in a 5′ to 3′ direction.
When an incorrect nucleotide is incorporated, it creates a mismatch at the 3′ end of the growing strand. The polymerase can then “backtrack,” removing the erroneous nucleotide before continuing synthesis. This ensures errors are corrected in the opposite direction of DNA synthesis, making this specific directionality indispensable for its biological roles.
The Proofreading Powerhouse in DNA
The primary function of 3′-5′ exonuclease activity is its role as a proofreading mechanism during DNA replication. As DNA polymerases synthesize new DNA strands, they occasionally incorporate an incorrect nucleotide, leading to a mismatched base pair. When such an error occurs, the newly synthesized strand’s 3′ end, containing the mispaired nucleotide, is no longer a suitable template for further elongation.
The DNA polymerase pauses and shifts its activity from synthesizing DNA to its intrinsic 3′-5′ exonuclease function. This exonuclease activity then precisely excises the incorrectly added nucleotide from the 3′ end of the growing strand. Once the wrong nucleotide is removed, the polymerase can resume its normal 5′ to 3′ synthesis, incorporating the correct nucleotide and continuing replication with high fidelity. This real-time error correction significantly improves the accuracy of DNA replication, reducing the error rate from approximately 1 in 10,000 to 1 in 100 million bases.
Beyond Replication DNA Repair
Beyond its immediate role in proofreading during replication, 3′-5′ exonuclease activity also participates in other distinct DNA repair pathways. These pathways address DNA damage and errors that may escape the initial proofreading step or arise from various environmental factors and spontaneous chemical changes.
For instance, some apurinic/apyrimidinic (AP) endonucleases possess 3′-5′ exonuclease activity. These enzymes can remove damaged or mismatched bases from the 3′ termini of DNA duplexes. In nucleotide incision repair (NIR), an AP endonuclease makes a cut on one side of a damaged nucleotide and then uses 3′-5′ exonuclease activity to expand the resulting single-strand break into a gap. This broader involvement highlights the versatility of 3′-5′ exonuclease activity in maintaining genome stability. The activity thus contributes to the overall cellular defense against genomic instability.
When Things Go Wrong Consequences of Errors
When 3′-5′ exonuclease activity is impaired or absent, the consequences for a cell can be serious, leading to an increased rate of mutations within the DNA. This deficiency means that errors made during DNA replication are not efficiently corrected, allowing misincorporated nucleotides to remain in the genetic sequence. The resulting accumulation of mutations can have profound downstream effects, including cellular dysfunction and widespread genomic instability.
Such increased mutation rates are linked to a higher risk of diseases, especially cancer. Mutations can affect important genes, like tumor suppressor genes or oncogenes, disrupting their normal function and contributing to uncontrolled cell growth and division. For example, mutations in the exonuclease domains of DNA polymerases delta (POLD) and epsilon (POLE) have been found to increase mutation rates and cancer risk, particularly in colorectal and endometrial cancers. These defects can lead to chronic replication stress, where the cell constantly attempts to repair accumulating errors, further exacerbating genomic instability and potentially driving disease progression.