Fluorescent Probes to Investigate Homologous Recombination Dynamics

• Main Author: Davenport, Eric Parker
• Format: Others
• Published: DigitalCommons@USU 2016
• Subjects: DNA Repair; Unnatural Amino Acid Incorporation; 4-azido-l-phenylalanin; Rad51; SSB; Chemistry; Organic Chemistry
• Online Access: https://digitalcommons.usu.edu/etd/5007; https://digitalcommons.usu.edu/cgi/viewcontent.cgi?article=6041&context=etd

There are multiple mechanisms by which DNA can become damaged. Such damage must be repaired for the cell to avoid ill-health consequences. Homologous recombination (HR) is a means of repairing one specific type of damage, a double-strand break (DSB). This complex pathway includes the Rad51-DNA nucleoprotein filament as its primary machinery. Current methodology for studying HR proteins includes the use of fluorescently labeled DNA to probe for HR dynamics. This technique limits the number of proteins that can be involved in experimentation, and often only works as an end reporter. The work here aims at improving upon standard techniques by creating two fluorescent protein probes. The first probe was developed by directly attaching a fluorophore to Saccharomyces cerevisiae Rad51 with the use of click chemistry and the incorporation of unnatural amino acids. This probe could function as a primary reporter on the formation and dissociation of the Rad51-DNA filament in the presence of pro- and anti- HR mediator proteins. The second probe was created by labeling the exterior cysteine residues of Plasmodium falciparum single strand DNA binding protein (SSB) with a fluorophore via maleimide chemistry. This probe acts as a secondary reporter for HR dynamics by signaling for when free single stranded DNA (ssDNA) is available.