DNA Polymerase I (Pol I) was first isolated from the bacterium Escherichia coli (E. coli). Its discovery in 1956 by Arthur Kornberg confirmed the existence of a cellular mechanism for DNA synthesis, establishing Pol I as the first known DNA polymerase. Pol I is a single polypeptide chain in E. coli that performs multiple functions, acting as a maintenance tool for the prokaryotic genome. This enzyme is fundamental in maintaining the integrity of the bacterial chromosome, primarily through its roles in replication finishing and various repair pathways.
The Three Enzymatic Activities
The diverse functions of DNA Polymerase I are made possible by its three distinct enzymatic activities. The primary role is its 5′ to 3′ polymerase activity, which catalyzes the addition of deoxyribonucleotides to the 3′-hydroxyl end of a growing DNA strand. This function requires a template strand and a pre-existing primer to extend. This synthesis activity is used specifically to fill in gaps within a DNA duplex, a process often termed “gap filling.”
Pol I also possesses a 3′ to 5′ exonuclease activity, which acts as a proofreading mechanism. If the enzyme mistakenly incorporates an incorrect nucleotide, this activity allows Pol I to pause, reverse direction, and excise the newly added, mismatched base from the 3′ end of the growing strand. This proofreading significantly enhances the fidelity of DNA replication and repair.
What truly sets Pol I apart is its unique 5′ to 3′ exonuclease activity. This domain allows the enzyme to degrade a DNA or RNA strand ahead of it, moving in the same direction as its synthesis activity. This ability to simultaneously remove nucleotides from the 5′ end of one strand and add nucleotides to the 3′ end of the adjacent strand is known as nick translation.
Primary Role in DNA Replication
The most recognized function of DNA Polymerase I is its role on the lagging strand, which is synthesized discontinuously in short segments called Okazaki fragments. DNA synthesis requires short RNA primers to begin, and these RNA segments must be removed and replaced with DNA to create a continuous double helix.
Pol I is recruited to the end of a newly synthesized Okazaki fragment. It uses its unique 5′ to 3′ exonuclease activity to degrade the RNA primer of the fragment ahead of it. As the RNA is removed nucleotide by nucleotide, the 5′ to 3′ polymerase activity immediately fills the resulting gap with deoxyribonucleotides.
The process continues until all the RNA nucleotides have been replaced by DNA, leaving a small break, or nick, in the DNA backbone between the completed Okazaki fragment and the one Pol I just finished filling. This final phosphodiester bond is then sealed by DNA ligase, which connects the two fragments into one continuous DNA strand.
Function in DNA Repair Pathways
Beyond its direct involvement in replication, DNA Polymerase I is a frequent participant in several pathways designed to correct damage in the bacterial genome. Pol I plays a role in Base Excision Repair (BER), which addresses small, non-helix-distorting base lesions like those caused by oxidation or deamination.
In BER, a damaged base is first removed by a specific DNA glycosylase, creating an abasic site in the DNA. Following this, other enzymes nick the DNA backbone, preparing a small gap that Pol I then recognizes. Pol I uses its 5′ to 3′ polymerase activity to insert the correct nucleotide(s) into this small gap, using the opposing strand as a template.
Pol I is also implicated in Nucleotide Excision Repair (NER), a mechanism that deals with bulky, helix-distorting lesions, such as those caused by ultraviolet light. After the damaged DNA segment is excised as a short oligonucleotide, Pol I steps in to synthesize the missing patch of DNA. In both repair contexts, Pol I’s ability to synthesize new DNA and its proofreading function ensure that the integrity and accuracy of the genetic sequence are restored following damage removal.