Initially studying for a BSc in Chemistry with Medicinal Chemistry I quickly found myself more interested in the more ‘biological side of things’. Pursuing this I started an MPhil in Biochemistry with Medicinal Chemistry and was introduced to the expression, purification and characterisation of proteins ultimately deciding this was the type of research I was passionate about.
Mini-chromosome maintenance (MCM) proteins are an integral part of the DNA replication process in archaea and eukaryotes where they serve as the replicative DNA helicase. The homohexameric MCMs found in archaea have proven to be a valuable model of eukaryotic heterohexameric MCM complexes. Archaea have been found in almost all environmental niches, living in extremes of temperatures, salt and pressurised conditions.
My PhD project aims to express, purify and characterize novel MCMs proteins found in archaea that inhabit a variety of environmental niches to be used as DNA motor proteins for Nanopore DNA sequencing. Initially, 20-30 MCMs will be subjected to expression trials and crude-scale activity assays to identify those with interesting properties. I will then select promising targets for more detailed functional and structural characterisation, which will include solving 3D structures of MCMs with and without DNA. I will use directed evolution approaches to enhance desirable properties of the MCMs.
I will also explore the role of cofactor proteins and their role in MCM oligomerisation, ATP hydrolysis, and DNA binding and unwinding. Ultimately, this work will reveal mechanistic insight into MCM assembly, substrate binding and helicase activity, and inform on how the homohexameric assemblies in archaea evolved into heterohexameric complexes in eukaryotes. By summer 2022, I will have screened a catalogue of uncharacterised archaeal MCMs, identified candidates to pursue, and started structural and functional studies.