Engineering DNA polymerase delta to make its activity Ca2+ dependent
Synthetic Biology Engineering Evolution Design SEED
2016
2016 Synthetic Biology: Engineering, Evolution & Design (SEED)
Poster Session
Accepted Posters
We are developing a DNA polymerase-based “molecular ticker-tape” for generating spatially and temporally resolved calcium ion concentration recordings in neurons. Calcium ion concentration changes are used as a proxy for neural firing. Such a “ticker-tape”, with its DNA synthesis error rate dependent on Ca2+ ion concentrations, would write the temporal trace of neural activity as error patterns onto DNA molecules making it possible to uncover neural interactions with single neuron precision. Two approaches are being attempted; direct tweaking of DNA polymerases to make their activity Ca2+ dependent and a modular approach where a DNA polymerase accessory protein is being made Ca2+ dependent. Proliferating cell nuclear antigen (PCNA) is a ring-shaped homotrimeric accessory protein that slides onto linear DNA and interacts with DNA polymerases, increasing their processivity at the cost of fidelity. S. cerevisiae polymerase delta (Pol delta) suffers approximately 2 to 4 fold loss in fidelity in vitro upon associating with its PCNA. Since a nontrimeric PCNA cannot interact with DNA polymerases we have made its trimerization Ca2+-dependent, by rationally fusing it with calmodulin (CaM). We are now studying the effect of Ca2+ on PCNA-CaM fusion and Pol delta interactions. Using the same rational approach we plan to create several Pol delta and calmodulin fusions. These fusions will be screened through an established S. cerevisiae plasmid-shuffling scheme, where only a functional DNA polymerase fusion will result in cell survival.