DNA replication is a fundamental process in all living organisms. It is a semi- discontinuous process in which the leading strand is synthesized continuously and the lagging strand is synthesized discontinuously as short Okazaki fragments (OF). The initiation of DNA synthesis requires DNA polymerase α (Pol α/primase) in complex with the primase to form a complex of four subunits. Pol α/primase is the only enzyme that can perform de novo DNA synthesis on single-stranded DNA. The catalytic subunit of the primase (PRIM1) synthesizes RNA primers that are approximately nine nucleotides long. The synthesized RNA primers are then passed intramolecularly to the polymerase active site (POLA1), which is thought to be mediated by the C-terminal domain of the primase large subunit (PRIM2-C) to synthesize dNTPs of approximately 20 nucleotides. The aim of this project was to optimize the expression and purification of Pol α/primase. The insect codon optimized POLA1 was C-terminally Strep tagged and transposed into the baculovirus genome. The other subunits of Pol α/primase, POLA2, PRIM1 and PRIM2 were cloned and expressed in E. coli cells. The cell lysates from Sf9 insect cells and E. coli cells were then mixed and purified by immunoaffinity chromatography and size-exclusion chromatography. This helped us achieve a pure Pol α/primase containing all the four subunits with a good total yield. The identity of all the protein bands were verified by mass spectroscopy. Furthermore, the protein demonstrated primer extension activity on multiple primer/template substrates. We also characterized the effect of the human replication protein A (RPA) on the DNA polymerization activity of Pol α/primase.
|Date of Award||Apr 2021|
|Original language||English (US)|
- Biological, Environmental Science and Engineering
|Supervisor||Samir Hamdan (Supervisor)|
- DNA Polymerase alpha/primase