- DNA repair mechanisms
- Regulated gene expression
- Protein characterization
- Microbial metabolism/resistance
- Molecular cloning
- Recombinant protein expression in bacteria
- Affinity chromatography
- Digital droplet PCR
- Western blot
- Yeast two-hybrid
- Fluorescence polarization
- Fluorescence microscopy
- Directed evolution
- Genomic sequencing
Despite being the most studied organism in the world, the E. coli genome still contains many genes with unknown functions. A recent screen for genes needed for recovery from ionizing radiation damage identified eight previously uncharacterized genes as having important functions in the repair of radiation-induced damage (Byrne et al, 2014). One of these genes, radD (previously yejH), is the focus of my research.
Characterization of radD is focused on both in vivo expression profiling and in vitro protein assays. Unlike most genes in the cell, the radD gene is predicted to be under the control of the σ54 promoter, a stress-specific transcriptional regulator involving binding of the rpoN subunit of RNA polymerase upstream of the gene. Using purified RpoN protein and segments of the radD promoter DNA, a student will characterize binding of the subunit to this genomic regulatory element, potentially documenting the first incidence of the σ54 promoter responding to DNA damage. This will be followed up by in vivo studies involving purifying the radD mRNA and using a digital droplet PCR to quantify the mRNA levels under various conditions.
A second focus of my research is the function of the isolated RadD protein. RadD contains the conserved domains of a superfamily 2 helicase, although no helicase activity has been observed in vitro, and the protein directly interacts with SSB, a central organizer of DNA repair functions. A student will use atomic force microscopy (AFM) through the Analytical Instrumentation Facility on campus to directly visualize RadD and SSB binding to DNA substrates.
Students involved in this research will contribute to the broad understanding of microbial metabolic pathways, particularly DNA repair, while refining their research and communication skills.