The maintenance of the integrity of DNA is crucial for life as any change could result in cellular abnormalities leading to disease. DNA is most stable as a double helix. However, many processes manipulating DNA require the presence of vulnerable single-stranded DNA (ssDNA). ssDNA-binding proteins (SSBs) have the ability to bind to ssDNA, stabilize it and thus allow DNA transactions to take place. Prokaryotic SSBs, found in bacteria and viruses that infect them, are comprised of a DNA-binding body and a negatively-charged flexible C-terminal tail. The removal of the tail results in increased ability of the protein to bind ssDNA. The goal of this project is to dissect the roles of the flexibility and negative charge of the tail for biological function using gp2.5, the ssDNA-binding protein of bacteriophage T7, as a prototype for prokaryotic SSB.
The natural form of gp2.5 and a mutant lacking the C-terminal tail have been successfully expressed in BL21 (DE3) E.coli cells and purified using Histagged technology. The generation of a mutant with an uncharged tail is currently in progress. Once this mutant protein is produced, the ssDNA binding abilities of all three versions of gp2.5 will be evaluated in order to better understand how SSBs and other proteins with flexible charged tails work. Overall, this study has the potential to contribute to selection of antibacterial agents that kill bacteria by disrupting the function of their ssDNA-binding proteins.
Molecular Dissection of the Mechanism of ssDNA-binding Proteins.
Undergraduate Review, 10, 25-30.
Available at: http://vc.bridgew.edu/undergrad_rev/vol10/iss1/10
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