This summer, I had the opportunity to do a nine-week placement in the research laboratory of Professor Anne Donaldson and Dr Shin-ichiro Hiraga at the Institute of Medical Sciences in Aberdeen. I had become interested in DNA replication and repair while doing additional reading for my Genetics degree, and I was eager to gain experience in a laboratory that explores the molecular basis of these processes. My interest attracted me to research carried out by the Donaldson-Hiraga group, which is focused on different DNA replication and repair proteins, the primary one being RIF1.
RIF1 is a protein present within the nucleus, which plays an important role in DNA replication and repair, as well as in chromatin structure. It was initially discovered in yeast and has since been identified in many other eukaryotic organisms, including humans. My project employed a biochemical approach called BPA crosslinking to explore the interaction between yeast RIF1 and a protein complex called the MCM complex. As eukaryotic DNA has two strands, they must first be separated or unwound prior to DNA replication, and the MCM complex does exactly this. When the MCM complex has phosphate groups added to it by another enzyme, it is able to unwind the two strands of DNA, subsequently allowing the initiation of DNA replication. However, RIF1 counteracts this process by facilitating the dephosphorylation of the complex, thereby performing a vital role in regulating replication timing. Yet, the MCM complex consists of multiple subunits, and despite its interaction with RIF1 being documented for years, it is not fully clear which precise regions of RIF1 interact with the different MCM subunits. My project aimed to address this.
BPA crosslinking is a fascinating approach. It involves the addition of BPA, a type of unnatural amino acid, into the amino acid sequence of a protein (such as RIF1). This approach allows the creation of different mutant proteins containing BPA at specific positions, and when these mutants are exposed to UV light, the inserted BPA crosslinks each mutant with interacting partners. Successful crosslinking can then be identified by a molecular biology technique called SDS-PAGE, and the precise interacting segments of a protein can be identified.
During my project, I was able to perform numerous laboratory techniques for the first time. I prepared different solutions, performed mutagenic and colony PCR, cultured and transformed both yeast and bacterial cells, and had the opportunity to carry out western blots and co-immunoprecipitation. My experiments worked, and as expected, specific regions of RIF1 that interact with the subunits of the MCM complex were identified. Furthermore, regions of RIF1 that interact with other proteins were also discovered, and the next step is to elucidate the identity of these proteins using mass spectrometry. Understanding the precise RIF1-MCM interaction in yeast could further clarify how DNA replication initiation occurs in humans, which in turn, could have implications in cancer research. In addition to being able to gain hands-on experience, I even had the chance to attend lab meetings and observe a confocal microscopy session. Thanks to my summer placement, I feel much more confident when carrying out laboratory techniques, and I am excited to apply my skills further.
Soon, I will begin the final year of my degree at the 51cg, and I hope to study a Masters in Molecular Medicine here. I am especially grateful for the support that I received from Professor Anne Donaldson and Dr Shin-ichiro Hiraga during my summer placement. I am also very grateful to have received funding from HotStart, which allowed me to contribute to a meaningful research project and meet new people along the way!
