DNA Polymerase I (Pol I) is an enzyme central to the final stages of DNA replication in prokaryotic organisms, such as E. coli. It was the first DNA polymerase discovered and plays a role secondary to the main replicative enzyme, DNA Polymerase III, which handles the bulk of new DNA synthesis. Pol I is unique because it performs three separate enzymatic actions within a single polypeptide chain: degradation, synthesis, and proofreading. These coordinated activities resolve a structural problem that arises during the synthesis of one of the two new DNA strands. Pol I acts as a cleanup and finishing tool, ensuring the newly replicated genome is complete and accurate.
Setting the Stage for DNA Polymerase I
DNA replication occurs at the replication fork, where the double helix is unwound. Because DNA polymerases can only synthesize new DNA by adding nucleotides to the 3’ end of a growing chain, the two template strands are copied differently. The leading strand is synthesized continuously. The lagging strand, however, must be synthesized discontinuously, in small segments called Okazaki fragments.
Each Okazaki fragment begins with a short RNA primer laid down by primase. DNA Polymerase III then extends this primer with DNA nucleotides. When Pol III finishes a fragment, it stalls at the 5’ end of the preceding fragment’s RNA primer, leaving a series of DNA fragments separated by short RNA sequences that must be removed and replaced with DNA.
The Primary Task: Removing RNA Primers
DNA Polymerase I’s most distinctive function is its 5’ to 3’ exonuclease activity. This activity allows the enzyme to recognize and degrade the RNA primers that initiated each Okazaki fragment. The 5’ to 3’ exonuclease acts by cleaving nucleotides one by one from the 5’ end of a nucleic acid chain, moving toward the 3’ end.
This degradation action eliminates the RNA nucleotides from the newly synthesized strand. Pol I approaches the junction where the DNA of one Okazaki fragment meets the RNA of the next, and its exonuclease domain starts chipping away at the RNA primer.
The presence of this 5’ to 3’ exonuclease activity is unique among the E. coli polymerases. The ability to simultaneously degrade the RNA ahead of it while synthesizing new DNA is referred to as nick translation. This primer removal function is necessary for completing the lagging strand and is required for cell viability in many bacteria.
Filling the Gaps in the Lagging Strand
Immediately following the removal of RNA nucleotides, DNA Polymerase I uses its 5’ to 3’ polymerase activity to synthesize new DNA, filling the gap it just created. This is a tightly coupled process where the enzyme does not fully detach, but rather moves along the DNA, synthesizing as it degrades. The polymerase domain requires a free 3’-hydroxyl (3’-OH) group to begin synthesis, which is provided by the 3’ end of the upstream Okazaki fragment.
The enzyme adds deoxyribonucleotides one at a time, ensuring correct base-pairing with the template strand. As the 5’ to 3’ exonuclease removes RNA from the front, the 5’ to 3’ polymerase inserts DNA at the back, extending the Okazaki fragment.
Once the RNA sequence is completely replaced, Pol I leaves a single-strand break, or nick, in the DNA backbone between the newly synthesized DNA and the next Okazaki fragment. This final nick is subsequently sealed by the separate enzyme, DNA ligase, ensuring the lagging strand becomes a single, continuous DNA molecule.
Ensuring Accuracy Through Proofreading
DNA Polymerase I also possesses a 3’ to 5’ exonuclease activity, which serves as a proofreading mechanism. This function allows the enzyme to check the nucleotides incorporated during the gap-filling process. The 3’ to 5’ exonuclease acts in the reverse direction of synthesis, removing nucleotides from the 3’ end of the growing strand.
If Pol I incorporates an incorrect nucleotide, the enzyme stalls. The 3’ to 5’ exonuclease then excises the mismatched nucleotide, allowing the polymerase to insert the correct base. This proofreading action contributes to the overall fidelity of the replication process.