Fluorescent Probes to Investigate Homologous Recombination Dynamics

As the average cell’s DNA undergoes roughly 1 million molecular lesions per day, learning about one of the repair mechanisms which fixes damaged DNA is a worthwhile endeavor. Such work may provide insights into how to treat or prevent diseases such as cancers and associated genetic disorders. Homologous recombination (HR) is one pathway which is used by cells to repair a specific type of DNA damage, double-strand breaks. While HR is a complex process involving over 25 different proteins, exactly how HR works, including its regulation, is largely unknown. One protein in particular, Rad51, forms a filament on the damaged DNA and is critical for the repair process.While current research often utilizes labeled DNA as a means of studying HR proteins, this work involves the development of two alternative means of studying HR. First of all, the Rad51 protein was itself tagged with a fluorophore with the use of a technique developed by Dr. Peter Schultz, which involves altering one of a protein’s amino acid residues to a synthetic, unnatural amino acid. The unnatural amino acid incorporated into Rad51 is then labeled with the addition of dye. This results in Rad51 being able to report through fluorescence that it is forming a filament with DNA. A second probe was made by taking single strand binding protein, which binds to single stranded DNA (ssDNA), and labeling the amino acid cysteine with a fluorophore. This probe works in studying HR by signaling when ssDNA becomes available.