Characterizing Binding Between Replication Protein A and the Human Shu Advanced

Background

DNA is topic to endogenous and exogenous sources of DNA injury. When DNA injury accumulates, DNA replication might be disrupted and end in cell demise. Quite a lot of DNA restore mechanisms have advanced in eukaryotes and prokaryotes to keep up genome stability. DNA restore pathways are extremely conserved between totally different species, nonetheless, they diverge within the proteins and protein complexes that carry out restore. One such conserved pathway is homologous recombination (HR) which has twin capabilities: each contributing to genetic range and repairing DNA injury through a template mechanism. Not too long ago, proteins which have canonically been outlined to operate in HR have been proven to moonlight at replication forks (Cong 2022). When replication forks stall, proteins are recruited to those DNA buildings and facilitate fork restart. Nonetheless, the precise mechanisms which can be concerned in fork restart have been the topic of curiosity on this final decade given the implication of replication stress in most cancers (Cox 2000, Cong 2022). Most cancers cells are extremely dividing and have an elevated variety of stalled replication forks.

My focus is the human Shu complicated, a protein complicated composed of SWSAP1 and SWS1 proteins. My mentor Dr. Hengel not too long ago discovered that the SWSAP1-SWS1 complicated interacts with different DNA restore proteins RAD51 and Replication Protein A (RPA). Importantly, we hypothesize that the SWSAP1-SWS1 and RPA interplay is stimulating templated restore by a HR protein referred to as RAD51. As Dr. Hengel was the primary one to determine the RPA and SWSAP1-SWS1 interplay; there’s nearly no information on how this interplay happens. RPA is the principle single-strand DNA binding protein in eukaryotes (also referred to as SSBPs) (Wold 1997; Spies 2013; Hengel, 2016). RPA is an important protein concerned in genome upkeep particularly replication, recombination, and restore. Inside DNA restore, RPA is concerned in all three pathways (homologous recombination, nucleotide excision restore, and mismatch restore), nonetheless, this challenge will probably be centered on RPAs position in homologous recombination.

Aim and Methodologies

To purify 3 totally different RPA constructs (RPA, alt-RPA and RPA 2+3) and use Protein Pulldowns and Förster resonance vitality switch (FRET) to raised perceive the best way by which RPA and Shu work collectively. 

Step one of this, that may probably take 2-3 weeks, will probably be purifying RPA and alt-RPA (I already expressed and grown up these samples throughout this previous spring semester). The second step will probably be to make a plasmid encoding RPA 2+3 from the unique RPA plasmid after which display the protein for expression. Then I’ll carry out protein pulldowns to determine if the three various kinds of RPA bind to Shu and if there’s time I’ll use FRET to discover how the presence of varied DNA substrates impacts this binding.

Understanding this protein interplay may inform the exploration of future therapies for reproductive cancers resembling breast and ovarian cancers.