About the Project
http://www2.mrc-lmb.cam.ac.uk/groups/jes/
The smooth progress of DNA replication is continuously challenged by problems on the DNA template. DNA damage has been the most intensively studied source of replication road blocks but, in addition, the very act of unwinding DNA allows some sequences to form secondary structures that also potently impede progression of the replication fork [1,2]. Further, recent work suggests that transient impediments formed by DNA secondary structures occur very frequently with the cell deploying multiple overlapping mechanisms to ensure the timely and complete replication of problematic sequences. This is important as failure to maintain processive replication at such sites can lead to both genetic and epigenetic instability, potentially fertile drivers of cancer development and evolution.
The project seeks to understand the molecular role played by specialised DNA polymerases in replicating genomic DNA sequences that form secondary structures [2]. In particular, it will focus on the role of the REV1 protein, which have previously demonstrated to play an important role in the replication of a common form of secondary structure known as the G quadruplex [3-5]. The precise molecular mechanism by which REV1 facilitates the replication of these structures remains elusive but is likely to involve both its DNA binding and catalytic activity as well as significant, but poorly understood, interactions with other factors involved in G quadruplex replication including specialised DNA helicases [4] and repriming activities [6].
The project will make use of innovative genetic and cell biological techniques that exploit local changes in the epigenome as a ‘lens’ to monitor delays in replication of specific structure-forming sequences in vivo in avian and human cells [1,4]. This will be combined with state-of-the art mass spectrometry methods to reveal the enzymatic networks that act to smooth the replication of ‘difficult’, structure-forming DNA sequences.
References
1. Šviković, S. and Sale J.E. (2017). The effects of replication stress on S phase histone management and epigenetic memory. J. Mol. Biol. 429, 2011-2029. doi:10.1016/j.jmb.2016.11.011.
2. Wickramasinghe, C.M., Arzouk, H., Frey, A., Maiter, A. & Sale, J.E. (2015). Contributions of the specialised DNA polymerases to replication of structured DNA. DNA Repair 23, 83-90. doi: 10.1016/j.dnarep.2015.01.004.
3. Sarkies, P., Reams, C., Simpson, L.J. and Sale, J.E. (2010). Epigenetic instability due to defective replication of structured DNA. Molecular Cell, 40, 703-713.
4. Sarkies, P., Murat, P., Phillips, L.G., Patel, K.J., Balasubramanian, S. and Sale, J.E. (2012). FANCJ coordinates two pathways that maintain epigenetic stability at G-quadruplex DNA. Nucleic Acids Research 40, 1485-1498.
5. Schiavone, D., Guilbaud, G., Murat, P., Papadopoulou, C., Sarkies, P., Prioleau, M.-N., Balasubramanian, S. and Sale, J.E. (2014). Determinants of G quadruplex-induced epigenetic instability in REV1-deficient cells. EMBO J. 33, 2507-20.
6. Schiavone, D., Jozwiakowski, S.K., Romanello, M., Guilbaud, G., Bailey, L.J., Sale, J.E. & Doherty, A.J. (2016). PrimPol is required for replicative tolerance of G quadruplexes in vertebrate cells. Molecular Cell 61, 161-169. doi: 10.1016/j.molcel.2015.10.038.
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