About me

I graduated with an upper second class honors degree in Biochemistry (BSc) from the University of Portsmouth. At this time, I joined the McGeehan group for my undergraduate research project. This primarily focused around optimising the expression and purification of a Cytochrome C reductase protein for X-ray crystallography.
Due to my enjoyment of research-based science, I decided to stay in Portsmouth and complete my masters of research (MRes) with Dr. Simon Kolstoes group, ultimately graduating with distinction. During my MRes, I expressed the Dengue and Zika envelope proteins in HEK293 mammalian cell lines. These target proteins were purified using metal ion and gel filtration chromatography. Finally, Surface plasmon resonance (SPR) was used to investigate the affinity binding of the Dengue and Zika envelope proteins to inhibitor compounds kindly isolated by our collaborators in Mexico.

My project

Fanconi anaemia (FA) is a genetic disease linked to insufficiently repaired DNA during replication. FA patients are susceptible to DNA crosslinking (ICL) resulting in an array of health defects including kidney failure and increased cancer susceptibility. ICL’s are identified by the FA pathway, a multi-protein repair complex that recruits downstream nucleases to unhook and remove the crosslink from the DNA duplex. Human Fanconi anaemia nuclease 1 (hFAN1) is a structure-specific exonuclease with an affinity for 5’ flaps and has been shown to function dependably and independently of the FA pathway. The structure of hFAN1 has been solved via X-ray crystallography however the underlying mechanism followed by kinetic studies still not fully understood.
Throughout my PhD in Professor Jane Grasby’s laboratory, I will express and purify hFAN1 using standardized bacterial expression systems accompanied by metal ion affinity, ion exchange, and gel-filtration chromatography. once purified, I will perform single and multiple turnover kinetics to gain quantitive data about hFAN1’s functionality and binding towards synthetically produced oligonucleotide substrates with varying 5′ flap lengths. Finally, I will perform FRET analysis to gain greater insight into the conformational dynamics of hFAN1 once bound to its target DNA substrates and investigate whether hFAN1 functions primarily in the monomeric or dimeric conformation.

Connect

LinkedIn: https://www.linkedin.com/in/reuben-ouanounou-426874104/