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Dr. Stefanie H. Chen

Dr. Stefanie H. Chen

Research Areas:

  • DNA repair mechanisms
  • Regulated gene expression
  • Protein characterization
  • Microbial metabolism/resistance

Techniques:

  • Molecular cloning
  • Recombinant protein expression in bacteria
  • Affinity chromatography
  • Digital droplet PCR
  • ChIP
  • SDS-PAGE
  • Western blot
  • Yeast two-hybrid
  • EMSA
  • Fluorescence polarization
  • Fluorescence microscopy
  • AFM
  • Directed evolution
  • Genomic sequencing

Project Description:

Despite being the most studied model 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 a Flag-tagged RpoN protein, a student will use ChIP-qPCR to observe 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 complemented by gene expression studies involving purifying the radD mRNA and using a digital droplet PCR to quantify the mRNA levels under various stress 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 purify wildtype RadD and several mutants to explore the functions of various domains of the protein, including ATP hydrolysis and binding to DNA and SSB.        

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.

References:

Byrne, R.T., S.H. Chen, E.A. Wood, E.L. Cabot, M.M. Cox. “Escherichia coli genes and pathways involved in surviving extreme exposure to ionizing radiation.” Journal of Bacteriology. 2014.
https://pubmed.ncbi.nlm.nih.gov/25049088/ 

Chen, S.H., R.T. Byrne, E.A. Wood, and M.M. Cox. “Escherichia coli radD (yejH) gene: a novel function involved in radiation resistance and double-strand break repair.” Molecular Microbiology. 2015.
https://www.ncbi.nlm.nih.gov/pubmed/25425430

Chen, S.H., R.T. Byrne-Nash, and M.M. Cox. “Escherichia coli RadD Protein Functionally Interacts with the Single-stranded DNA-binding Protein.” Journal of Biological Chemistry. 2016.
https://www.ncbi.nlm.nih.gov/pubmed/27519413

Osorio Garcia, M.A., K.A. Satyshur, M.M. Cox, and J.L. Keck. “X-ray crystal structure of the Escherichia coli RadD DNA repair protein bound to ADP reveals a novel zinc ribbon domain.” PLoS One. 2022.
https://pubmed.ncbi.nlm.nih.gov/35482735/ 

Shereda, R.D., A.G. Kozlov, T.M. Lohman, M.M. Cox, and J.L. Keck. “SSB as an organizer/mobilizer of genome maintenance complexes.” Critical Reviews in Biochemistry and Molecular Biology. 2006.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2583361/

Sun, Z., H.Y. Tan, P.R. Bianco, & Y.L. Lyubchenko. “Remodeling of RecG Helicase at the DNA Replication Fork by SSB.” Scientific Reports. 2015.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4894433/ 

Zhao, K., M. Liu, and R.R. Burgess. “Promoter and regulon analysis of nitrogen assimilation factor, σ54, reveal alternative strategy for E. coli MG1655 flagellar biosynthesis.” Nucleic Acids Research. 2010.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2831329/

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