- DNA Polymerase I is a DNA-dependent DNA polymerase first discovered in Escherichia coli by Arthur Kornberg in 1956.
- It was the first DNA polymerase to be identified and has since served as a cornerstone in understanding DNA replication and repair.
- Although it is not the main replicative enzyme in E. coli (a role performed by DNA Polymerase III), DNA Polymerase I plays a crucial role in DNA repair and lagging strand processing, particularly in the removal and replacement of RNA primers in Okazaki fragments.
- Structurally, DNA Polymerase I is a single polypeptide enzyme with three key enzymatic functions: a 5′→3′ DNA polymerase activity that adds nucleotides in a template-directed manner, a 3′→5′ exonuclease activity that performs proofreading by excising mismatched bases, and a 5′→3′ exonuclease activity that removes RNA primers or damaged nucleotides ahead of the polymerization site. This unique combination of activities enables the enzyme to synthesize DNA while simultaneously editing and replacing damaged or unwanted sequences.
- A modified form of the enzyme, known as the Klenow fragment, is produced by removing the 5′→3′ exonuclease domain. This fragment retains the polymerase and proofreading functions, making it highly useful in molecular biology applications. The Klenow fragment is commonly employed in blunt-end DNA synthesis, fill-in reactions of 5′ overhangs, and second-strand cDNA synthesis.
- In the cell, DNA Polymerase I primarily functions in DNA repair pathways such as base excision repair and nucleotide excision repair, as well as in the processing of lagging strand DNA during replication. It replaces RNA primers with DNA and ensures the integrity of the genome by filling in short gaps after removal of damaged sequences.
- In biotechnology, DNA Polymerase I and its derivatives are widely used in techniques like nick translation, DNA labeling, site-directed mutagenesis, and strand displacement synthesis. Its high fidelity and robust activity under mild conditions make it a versatile tool for many DNA manipulation protocols.
- In conclusion, E. coli DNA Polymerase I is a multifunctional enzyme essential for maintaining genomic stability through its roles in repair and replication. Its broad utility in research and biotechnology underscores its continued importance in molecular biology.
