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The functional assembly of the mammalian minichromosome maintenance complex

Hesketh, Emma Louise (2014) The functional assembly of the mammalian minichromosome maintenance complex. PhD thesis, University of York.

Available under License Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 UK: England & Wales.

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The minichromosome maintenance complex (MCM2-7) is the putative DNA helicase in eukaryotes, and is essential for DNA replication. Mis-regulation of its assembly and function can lead to genomic instability, replication stress and genome re-replication, which are powerful drivers toward the acquisition of mutations and the formation of cancer. I have produced and purified recombinant human MCM2-7 (hMCM) in Escherichia coli (E. coli). Recombinant hMCM has ATP hydrolysis and helicase activity, when assayed in the absence of post-translational modifications or accessory proteins. Using electron microscopy asymmetric single particle reconstruction, I have produced 23 Å resolution structures. The structures reveal that recombinant hMCM forms an asymmetric complex that changes conformation in the presence of a forked DNA substrate. By applying serial extractions to mammalian cells synchronised by release from quiescence, I have revealed dynamic changes in the sub-nuclear compartmentalisation of MCM2 as cells pass through late G1 and early S phase, identifying a brief window when MCM2 becomes transiently attached to the nuclear matrix. This suggests that functional MCM2-7 loading takes place at the nuclear matrix. Using this information I have developed a system to study the regulated assembly of recombinant hMCM, to support molecular dissection of the loading process. Using cellular extracts, I show regulated binding of recombinant hMCM, which translates into initiation of DNA replication and is stimulated by cyclin E/CDK2. Furthermore, I have demonstrated that both cyclin A/CDK2 and Dbf4/Drf1-dependant kinase (DDK) are required for inducing a conformational change in hMCM that is associated with S phase of the cell cycle. Understanding of the assembly of hMCM will serve as a foundation for analysis of corruption of the process, its effect on genome instability and gene expression, and how these events lead to the ‘birth’ of cancer cells. Very early events in the development of cancer cells are most likely to yield new and effective opportunities for detection and treatment of a broad range of cancers.

Item Type: Thesis (PhD)
Academic Units: The University of York > Biology (York)
Identification Number/EthosID: uk.bl.ethos.647070
Depositing User: Ms Emma Louise Hesketh
Date Deposited: 28 May 2015 15:05
Last Modified: 24 Jul 2018 15:20
URI: http://etheses.whiterose.ac.uk/id/eprint/8984

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