DNA polymerase I (also known as Pol I) is an enzyme involved in bacterial DNA replication. It was the first known DNA polymerase, discovered by Arthur Kornberg in 1956. (and the first known of any kind of polymerase). It was first discovered in E. coli and is now found in all prokaryotes. The E. coli Pol I enzyme has 928 amino acids and is a processive enzyme that can catalyse numerous polymerisation processes in a sequential manner without releasing the single-stranded template. Pol I’s major physiological purpose is to aid in the repair of damaged DNA, but it also helps to join Okazaki fragments by removing RNA primers and replacing them with DNA.
History
Arthur Kornberg developed DNA polymerase I in 1956. His preliminary findings were originally presented at the Federation of American Societies for Experimental Biology (FASEB) annual meeting in Atlantic City, New Jersey, in 1956. Reviewers of his first work urged that the authors use the term ‘polydeoxyribonucleotide’ instead of ‘DNA,’ but after an appeal to the editor-in-chief, John Edsall, the term ‘DNA’ was allowed (Friedberg 2006). In 1958, Lehman et al. and Bessman et al. released two further publications that proved DNA polymerase was responsible for DNA replication. In 1959, Kornberg was awarded the Nobel Prize for discovering DNA polymerase I. Jovin deduced the amino acid composition in 1969. DeLucia and Cairns identified an E. coli strain with a mutation affecting the DNA polymerase in the same year, and were surprised to discover that the mutant produced DNA regularly.
General Structure
Pol I is primarily involved in the repair of DNA that has been damaged. Pol I belongs to the alpha/beta protein superfamily, which includes proteins with -helices and -strands arranged in an irregular pattern. Multiple domains make up Pol I, which has three unique enzymatic functions. Thumb, finger, and palm domains are three domains that work together to keep DNA polymerase active. In a process termed as proofreading, an exonuclease active site located close to the palm domain eliminates improperly integrated nucleotides in a 3′ to 5′ orientation. Another exonuclease active site is found in the fifth domain, which removes DNA or RNA in a 5′ to 3′ direction and is required for RNA primer removal during DNA replication or DNA repair.
Function
Pol I has four different enzymatic activities:
- A DNA polymerase activity that is 5’3′ (forward) DNA dependent and requires a 3′ primer site and a template strand.
- Proofreading is mediated by a 3’→5′ (reverse) exonuclease activity.
- During DNA repair, nick translation is mediated by a 5’→3′ (forward) exonuclease activity.
- A DNA polymerase activity that is 5’→3′ (forward) RNA dependent. Pol I has a lesser efficiency (0.1–0.4%) while working with RNA templates than it does with DNA templates, and this activity is likely of modest biological consequence.
An experiment was undertaken with a defective Pol I mutant strain of E. coli to investigate whether Pol I was predominantly employed for DNA replication or DNA damage repair. A mutant strain lacking Pol I was identified and given a mutagen treatment. The mutant strain produced bacterial colonies that grew properly but lacked the protein Pol I. This proved that Pol I isn’t necessary for DNA replication. However, the mutant strain exhibited exceptional vulnerability to certain DNA-damaging agents, including as UV radiation. As a result, Pol I was found to be more likely to be involved in DNA damage repair than in DNA replication.
Mechanism
RNase H removes the RNA primer (produced by primase) from the lagging strand during replication, and then polymerase I fills in the missing nucleotides between the Okazaki fragments in a 5’3′ direction, proofreading for errors along the way. It’s a template-dependent enzyme, which means it only adds nucleotides that appropriately base pair with a template DNA strand. These nucleotides must be in the correct orientation and geometry to base pair with the DNA template strand so that DNA ligase can connect the fragments into a single strand of DNA. Different dNTPs can bind to the same active site on polymerase I, according to polymerase I research. Only after undergoing a conformational shift is Polymerase I capable of actively discriminating between distinct dNTPs. Pol I then checks the base pair generated by the bound dNTP and a matching base on the template strand for correct geometry and alignment.
Despite its early identification, polymerase I was quickly discovered to not be the enzyme responsible for most DNA synthesis—DNA replication in E. coli occurs at a rate of 1,000 nucleotides per second, whereas polymerase I only produces base pairs at a rate of 10 to 20 nucleotides per second. Furthermore, the fact that E. coli has just two replication forks did not correlate with its biological abundance of roughly 400 molecules per cell.
CONCLUSION
In a 5’3′ orientation, DNA Polymerase I catalyses the template-directed polymerization of nucleotides into duplex DNA. DNA Polymerase I has a 3’5′ exonuclease activity, which lowers the error rate during DNA replication, as well as a 5’3′ exonuclease activity, which allows the enzyme to replace nucleotides in the developing strand of DNA via nick translation. Purified from recombinant E. coli, the enzyme is capable of catalysing the de novo synthesis of synthetic homopolymers, making it a practical way to prepare a variety of specific DNA substrates.