Structural and functional characterisation of Mcb1 and the MCMᴹᶜᵇ¹ complex in Schizosaccharomyces pombe
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The MCM helicase plays an important role in eukaryotic DNA replication, unwinding double stranded DNA ahead of the replication fork. MCM is a hetero-hexamer consisting of the six related proteins, Mcm2-Mcm7. The distantly related MCM-binding protein (MCM-BP) was first identified in a screen for proteins interacting with MCM2-7 in human cells and was found to specifically interact with Mcm3-7 but not Mcm2. It is conserved in most eukaryotes and seems to play an important role in DNA replication but its exact function is not clear yet. This study contributes to the understanding of the fission yeast homologue of MCM-BP, named Mcb1, but also of MCM-BP in general. Results presented in this thesis document the initial biochemical characterisation of the complex Mcb1 forms with Mcm proteins, the MCMᴹᶜᵇ¹ complex. Interactions of Mcb1 with Mcm proteins, potential interaction sites between the proteins and the size of the complex were analysed using a variety of methods, including tandem affinity purification, co-immunoprecipitation, sucrose gradients and in vitro pull-down assays. Sequence analysis and structure prediction were utilised to gain some insight into Mcb1 and MCM-BP ancestry and structure. Results presented here indicate that fission yeast Mcb1 shares homology with Mcm proteins and forms a complex with Mcm3-Mcm7 but not Mcm2 and thus replaces the latter in an alternative high molecular weight complex that is likely to have an MCM-like appearance. Deletion of mcb1⁺ showed that Mcb1 is essential in fission yeast. To examine the cellular function of the protein, temperature-sensitive mutants were generated. Inactivation of Mcb1 leads to an increase in DNA damage and cell cycle arrest in G2-phase depending on the activation of the Chk1 dependent DNA damage checkpoint. Similar observations were made when Mcb1 was overexpressed, indicating that certain levels of the protein are important for accurate DNA replication. Construction of truncated versions of Mcb1 suggested that almost the full-length protein is needed for proper function.
Thesis, PhD Doctor of Philosophy
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