Background

DNA is subject to endogenous and exogenous sources of DNA damage. When DNA damage accumulates, DNA replication can be disrupted and result in cell death. A variety of DNA repair mechanisms have evolved in eukaryotes and prokaryotes to maintain genome stability. DNA repair pathways are highly conserved between different species, however, they diverge in the proteins and protein complexes that perform repair. One such conserved pathway is homologous recombination (HR) which has dual functions: both contributing to genetic diversity and repairing DNA damage via a template mechanism. Recently, proteins that have canonically been defined to function in HR have been shown to moonlight at replication forks (Cong 2022). When replication forks stall, proteins are recruited to these DNA structures and facilitate fork restart. However, the specific mechanisms that are involved in fork restart have been the subject of interest in this last decade given the implication of replication stress in cancer (Cox 2000, Cong 2022). Cancer cells are highly dividing and have an increased number of stalled replication forks.

My focus is the human Shu complex, a protein complex composed of SWSAP1 and SWS1 proteins. My mentor Dr. Hengel recently found that the SWSAP1-SWS1 complex interacts with other DNA repair proteins RAD51 and Replication Protein A (RPA). Importantly, we hypothesize that the SWSAP1-SWS1 and RPA interaction is stimulating templated repair by a HR protein called RAD51. As Dr. Hengel was the first one to identify the RPA and SWSAP1-SWS1 interaction; there is almost no knowledge on how this interaction occurs. RPA is the main single-strand DNA binding protein in eukaryotes (also known as SSBPs) (Wold 1997; Spies 2013; Hengel, 2016). RPA is an essential protein involved in genome maintenance specifically replication, recombination, and repair. Within DNA repair, RPA is involved in all three pathways (homologous recombination, nucleotide excision repair, and mismatch repair), however, this project will be focused on RPAs role in homologous recombination.

Goal and Methodologies

To purify 3 different RPA constructs (RPA, alt-RPA and RPA 2+3) and use Protein Pulldowns and Förster resonance energy transfer (FRET) to better understand the way in which RPA and Shu work together. 

The first step of this, that will likely take 2-3 weeks, will be purifying RPA and alt-RPA (I already expressed and grown up these samples during this past spring semester). The second step will be to make a plasmid encoding RPA 2+3 from the original RPA plasmid and then screen the protein for expression. Then I will perform protein pulldowns to identify if the three different types of RPA bind to Shu and if there is time I will use FRET to explore how the presence of various DNA substrates affects this binding.

Understanding this protein interaction could inform the exploration of future therapies for reproductive cancers such as breast and ovarian cancers.