Prof Neil Kad – University of Kent
Tim Fenton – University of Southampton
The human genome possesses 11 members of the APOBEC family of proteins, that are involved in a variety of roles from antibody diversification to mRNA editing. One role is of particular interest, APOBEC3A is capable of deaminating single stranded DNA, i.e. the conversion of cytosine to uracil. Such deamination events lead to a signature APOBEC pattern that is observed in numerous cancers.
This is likely caused by genome instability either through strand breaks as the cell attempts to remove the uracil, or mutation as the uracil is mistaken for thymine during replication, leading to a conversion from C to T.
Exactly where this occurs is unclear because DNA is double-stranded except during replication and transcription. However, prevailing evidence is that deamination occurs primarily on the lagging strand during DNA synthesis. Our goal in this project is to understand to what DNA structures APOBEC binds. The student will synthesize DNA constructs and study them using biochemical and biophysical approaches to learn how they affect the affinity of a fluorescently labelled APOBEC. In parallel, the student will suspend these structures between beads caught in laser tweezers so that a single APOBEC can be observed directly as it binds to the DNA and finds the target structure. We also hypothesize that APOBECs could be involved in the control of transcription through binding DNA structures formed by promoters, this highly exciting aspect of APOBEC function has not been observed to date and will be truly ground-breaking in its impact. To fully investigate this function the student will also study this in live cells using a variety of cell biological techniques. Our ultimate goal is to understand the mechanism of action of this highly enigmatic protein with clear roles in cancer as well as immunity from viral RNA (which it also deaminates).