Oncogene expression leads to DNA replication defects that drive tumour development. Replication defects are retained in many mature cancers, and a deeper understanding of the mechanisms and regulation of chromosome duplication will identify novel targets for future therapies. We study the large molecular machine known as the replisome, which is assembled and then disassembled just once per cell cycle, to ensure that cells make a single copy of each chromosome. We found that replisome disassembly is highly regulated during DNA replication termination and is initiated by ubiquitylation of the DNA helicase around which the replisome is built, leading to a disassembly reaction that requires the Cdc48 ‘segregase’ (Refs. 1-4). Excitingly, it now appears that cells also disassemble the replisome as the first step of specific DNA damage repair reactions, or as part of pathways that process regions of incomplete DNA replication during mitosis. Much remains to be learnt of the underlying mechanisms and regulation of these multiple pathways for replisome disassembly, a deeper understanding of which is likely to suggest new targets for future cancer therapeutics.
We are using a wide range of cutting-edge approaches to address these issues, ranging from yeast cell biology, in vitro reconstitution of replisome disassembly with purified proteins, the C. elegans early embryo as a model system with which to identify metazoan-specific aspects of replisome regulation, and mouse ES cells to study the regulation of the mammalian replisome.
A rotation project would provide experience in one of the following areas, guided by members of our group:
1. Reconstituting the events of DNA replication termination and replisome disassembly in vitro.
We have recently reconstituted replisome ubiquitylation and disassembly using replisomes purified from yeast cell extracts. The next step would be to generate recombinant replisomes from purified yeast proteins, to try and define which components are required for each step of the disassembly reaction in vitro.
2. Identifying ubiquitin ligases that drive novel replisome disassembly pathways during mitosis.
The student would use RNAi to screen candidate ubiquitin ligases in the C. elegans early embryo, to see which might be important for mitotic replisome disassembly in response to incomplete DNA replication.
3. Studying replisome disassembly in mouse ES cells
The student would isolate the replisome from mouse ES cells with tagged replisome subunits (made by CRISPR), and then screen by mass spectrometry for associated ubiquitin ligases.
1. Maric, M., Maculins, T., De Piccoli, G. and Labib, K. (2014). Cdc48 and a ubiquitin ligase drive disassembly of the CMG helicase at the end of DNA replication. Science, 346 (6208):1253596. Doi: 10.1126/science.1253596.
2. Maculins, T., Nkosi, P. J., Nishikawa, H. and Labib, K. (2015). Tethering of SCF to the Replisome Promotes Efficient Ubiquitylation and Disassembly of the CMG Helicase. Curr. Biol., 25, 2254-2259.
3. Sonneville R, Moreno SP, Knebel A, Johnson C, Hastie CJ, Gartner A, Gambus A, Labib K. (2017) CUL-2LRR-1 and UBXN-3 drive replisome disassembly during DNA replication termination and mitosis. Nat Cell Biol. 19, 468-479.
4. Maric M, Mukherjee P, Tatham MH, Hay R, Labib K. (2017) Ufd1-Npl4 Recruit Cdc48 for Disassembly of Ubiquitylated CMG Helicase at the End of Chromosome Replication. Cell Rep. 18, 3033-3042.