DNA Polymerase I (Pol I) is an enzyme that plays a role in DNA processes within prokaryotic cells, such as bacteria. Discovered in 1956, it was the first DNA polymerase identified. Its functions are diverse, contributing to both the copying of DNA and the repair of damaged DNA segments.
The Primary Roles of DNA Polymerase I
DNA Polymerase I exhibits three distinct enzymatic activities: 5’→3′ polymerase activity, 3’→5′ exonuclease activity, and 5’→3′ exonuclease activity. These activities allow it to perform various tasks related to DNA replication and repair. The 5’→3′ polymerase activity enables the enzyme to synthesize new DNA strands by adding nucleotides in a specific direction. This is particularly important for filling in gaps that remain after the removal of RNA primers during DNA replication or when repairing damaged DNA.
The 3’→5′ exonuclease activity functions as a proofreading mechanism. If an incorrect nucleotide is mistakenly incorporated into the growing DNA strand, this activity allows Pol I to detect and remove the mismatched base from the 3′ end. This proofreading capability significantly reduces the error rate during DNA synthesis, contributing to genetic stability.
The 5’→3′ exonuclease activity allows Pol I to degrade DNA or RNA from the 5′ end. It removes RNA primers that initiate DNA synthesis on the lagging strand during replication. As it removes these RNA primers, Pol I simultaneously fills the resulting gaps with DNA nucleotides, a process sometimes called nick translation. This dual action ensures that the newly synthesized DNA strand is continuous and free of RNA segments.
What Makes DNA Polymerase I Unique?
While other DNA polymerases are involved in DNA synthesis, DNA Polymerase I stands out due to its specific combination of enzymatic activities. Most DNA polymerases possess 5’→3′ polymerase activity for synthesizing DNA and 3’→5′ exonuclease activity for proofreading. However, Pol I is unique among bacterial polymerases in also having a 5’→3′ exonuclease activity.
This 5’→3′ exonuclease activity is important for primer removal and DNA repair. Other polymerases, such as DNA Polymerase III, are the primary enzymes for extensive DNA synthesis during replication. Pol I’s capabilities allow it to handle specific clean-up and repair tasks that other polymerases do not perform.
Using DNA Polymerase I in Research
The distinct properties of DNA Polymerase I have made it a valuable tool in molecular biology research. One significant application involves a modified version of the enzyme known as the Klenow fragment. This fragment is produced by treating Pol I with a protease, which cleaves the enzyme into two parts. The larger part, the Klenow fragment, retains the 5’→3′ polymerase activity and the 3’→5′ exonuclease (proofreading) activity but lacks the 5’→3′ exonuclease activity.
The Klenow fragment’s ability to synthesize DNA and proofread without 5′ end degradation makes it useful for laboratory procedures. It is commonly used for synthesizing double-stranded DNA from single-stranded templates, filling in recessed 3′ ends of DNA fragments to create blunt ends, and preparing radioactive DNA probes. It is also used in DNA sequencing methods and for synthesizing the second strand of cDNA.