Structural Biochemistry/DNA polymerase I

DNA polymerase I (E.Coli) is an enzyme that fills gaps in duplexes by stepwise addition of nucleotides to 3'-ends for DNA sequence. DNA polymerase I is an important enzyme to be used in DNA sequencing, DNA replication, and PCR. It is used to insert the right nucleotides into a primer to start base-pairings.

Robert Lehman studied DNA polymerase early on in his career under a lab in St. Louis in 1955. What triggered his interest was the discovery of DNA of the T-even phages that contained hydroxymethcytosine instead of cytosine and he was eager to determine how this hydroxymethylcytosine was made. He found that extracts of T2-infected E. coli did incorporate this carbon 14 label into dCMP so he decided to purify this enzyme that was creating the activity. A colleague of his at the time found that carbon 14 labeled thymidine incorporated into an acid-insoluble product by E.coli which demonstrated that there was DNA synthesis in vetro the cell. They found out that E.coli converted thymidine in the presence of ATP into thymidine-X which was later identified as dTMP and this activity was thymidine kinase. Kinase is a catalytic activity where an phosphate group is added onto a nucleotide. They determined after experimentation that incubation of the precipitate the activity increased which demonstrated that more than one enzyme was required for the incorporation of dTTP into an acid-insoluble product. After they purified each of the deoxynucleotide kinase, they discovered and characterized the four dNTPs as dTTP, dCTP,dGTP, and dATP. It was determined that all dNTP were needed for high activity. We now know that DNA polymerase requires a primer strand to begin the DNA chain replication and is not able to just begin replication out of nowhere and that in order to synthesize DNA, it required a DNA primer, a template, and all four dNTPs which allowed the DNA polymerase to synthesize DNA. If anything was missing, activity would slow down. The DNA polymerase discovered by Robert Lehman and his team is now known as DNA polymerase I. DNA polymerase I plays an essential role in processing the Okazaki fragments produced during the replication on the lagging strand during DNA replication at the replication fork that connects the fragments. Later on in his career, he went back to studying DNA polymerase and it was determined that the essential role of the Polymerase I in DNA replication is the five prime to three prime exonuclease that takes out this RNA primer and initiates the Okazaki fragment synthesis which then triggers the filling in of the gaps before becoming joined by the DNA ligase.