This Administrative Equipment Supplement is submitted jointly with my University at Buffalo, SUNY, Department of Biochemistry colleague, Dr. David Fernando Estrada (R35 GM133375). This supplement request is attached to my parent R01 grant GM130761, entitled ?Coordination of high fidelity replication with mutagenic translesion synthesis,? for which I am lead PI. The purpose of this Administrative Supplement is to requested 50% of the funds required to purchase a FortBio OctetRed96e Bio-Layer Interferometry (BLI) instrument for rapid and reliable kinetic analysis of macromolecular interactions in real time without the need for substrate labelling. This instrument has several advantages over Surface Plasmon Resonance (SPR), and is desperately needed to replace our currently unusable Reichert SPR instrument. Although our Reichert SPR instrument was functional when the parent R01 grant was submitted, it has since become unusable. This proposal has the full support of my School?s Dean and my Department Chair, which together are providing 50% of the total cost for the OctetRed96e, including costs for training. This FortBio OctetRed96e BLI instrument is essential for the goals of the parent R01 grant as all three aims heavily rely on a rapid and reliable method for kinetic analysis of protein- protein interactions, particularly those involving wild type and mutant partners. We recently had the opportunity to demo the OctetRed96e in our own lab using our own proteins. During this time, we confirmed unambiguously that it was ideally suited for analyzing the exact types of protein-protein interactions required in our parent R01 grant. Of particular note, the OctetRed96e will analyze these interactions ~64-times faster and at ~3% the costs of our unusable Reichert SPR instrument. As a direct result of this significantly increased capacity and reduced cost, we will be able to explore directions not possible with our currently unusable Reichert SPR instrument. In summary, the acquisition of the FortBio OctetRed96e will restore our ability to analyze the kinetics of discrete protein-protein interactions at a significantly increased capacity and reduced cost. As a result, it will open up several new research directions that were not previously possible using our Reichert SPR instrument.
Mutations contribute directly to human diseases, including cancers; they also complicate treatment of individuals infected with microbial pathogens by conferring antibiotic resistance and adaptation of these pathogens to their human host. We will define mechanisms by which low fidelity E. coli DNA polymerases gain access to the replication fork to catalyze mutations. We anticipate that our results will identify critical features of these mechanisms that can be targeted to control bacterial replication and its fidelity for therapeutic gain, and will serve as a framework for understanding similar control networks in other organisms, including humans.
