About the Project
During the eukaryotic cell division cycle, the genome must be precisely duplicated with no sections left unreplicated and no sections replicated more than once. Failure to do this causes major genome abnormalities including deletion and amplifications, which can drive a range of pathologies such as cancer. We have recently provided evidence for a new pathway allowing cells with incompletely replicated genomes to undergo cell division, allowing daughter cells to complete replication. This PhD project will use a combination of experimental and theoretical approaches to explore this novel pathway.
The eukaryotic cell cycle is divided into two non-overlapping phases: during mitosis and G1 replication origins are “licensed” by being loaded with the ’Mini-Chromosome Maintenance’ (MCM2-7) proteins, and then during S phase these licensed replication origins are transformed into replication forks which perform the copying process. Replication forks can stall irreversibly, and if two converging forks stall with no intervening licensed origin - a “double fork stall” - replication cannot be completed by conventional means. We have recently shown that in organisms such as humans with large (gigabase-sized) genomes, a few stretches of DNA typically remain unreplicated when cells enter mitosis and are segregated to daughter cells via structures called ultrafine anaphase bridges. In these daughter cells a protein called 53BP1 then binds to these inherited DNA structures.
The PhD project will explore the mechanism that allows unreplicated DNA to be segregated to daughter cells, and will test a number of specific hypotheses about how it works. First, we will examine the role of DNA repair pathways (such as the Fanconi Anaemia pathway) in protecting unreplicated DNA from being degraded before cells enter mitosis. Second, we will examine how cells solve the challenge of unwinding the two unreplicated template strands so that they can be segregated to daughter cells and will investigate whether mitotic chromosome condensation provides the force to do this. Third, we will examine the role of 53BP1 in protecting these structures during G1, and will test the idea that one of 53BP1’s main roles is to prevent inappropriate recombination events in G1.
The project will use a combination of experimental and theoretical approaches, and will build on a successful collaboration that has been established between Prof Blow (experimental) and Prof Newman (theoretical). Experimental work will involve cell biology approaches using human tissue culture cells (including immunofluorescence, FACS, siRNA, CRISPR/CAS) and biochemical approaches (immunoblotting, immunoprecipitation, recombinant protein work) using Xenopus (frog) egg extracts. Theoretical work will involve a range of mathematical and physical modelling approaches to analyse the consequences of fork stalling. The project would suit students with a biological background but who have an interest in theoretical modelling.
References
Newman, T.J., Mamun, M.A., Nieduszynski, CA. and Blow, J.J. (2013). Replisome stall events have shaped the distribution of replication origins in the genomes of yeasts. Nucleic Acids Res. 41, 9705-9718.
Moreno, A., Carrington, J.T., Albergante, L., Al Mamum, M., Haagensen, E.J., Komseli, E.-S., Gourgolis, V.G., Newman, T.J. and Blow, J.J. Unreplicated DNA remaining from unperturbed S phases passes through mitosis for resolution in daughter cells. Proc Natl Acad Sci USA, in press.
Al Mamum, M., Albergante, L., Moreno, A., Carrington, J.T., Blow, J.J. and Newman, T.J. Inevitability and containment of replication errors for eukaryotic genome lengths spanning Megabase to Gigabase. Proc Natl Acad Sci USA, in press.
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