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Title: Biochemical characterisation of pre-replicative complex architecture
Author: Mehanna, A.
ISNI:       0000 0004 5362 9259
Awarding Body: University College London (University of London)
Current Institution: University College London (University of London)
Date of Award: 2014
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Pre-replicative complexes (pre-RCs), containing the helicase Mcm2-7, are assembled on origins of replication during G1 phase of the cell cycle. This ‘licenses’ origins for subsequent activation during S-phase. The loading of the Mcm2-7 complex requires ATP hydrolysis and the licensing factors ORC, Cdc6 and Cdt1, and results in the assembly of a head-to-head double hexamer of Mcm2-7 bound around duplex DNA. To understand how the Mcm2-7 complex is loaded into a double hexamer, we need a better understanding of the stoichiometry and positioning of licensing factors relative to each other during pre-RC assembly. To address this, I used a tagging and immunoaffinity purification strategy. For this purpose, I generated purified protein preparations where subunits of the licensing proteins were fused to either a 9x Myc or a 3x FLAG tag. These proteins were tested for their ability to support loading of the Mcm2-7 complex in vitro. I used the tagged proteins in an established in vitro pre-RC assembly assay coupled with an immunoaffinity purification approach. I found that in the absence of ATP hydrolysis, one molecule each of ORC, Cdc6 and Cdt1 recruit a single Mcm2- 7 hexamer to origin DNA. Using an ATPase mutant, I showed that ATP hydrolysis by Cdc6 is not required for Mcm2-7 double hexamer formation. I found that a conserved C-terminal region of Mcm3 is critical for Mcm2-7 recruitment to ORCCdc6- DNA. Mutations in this C-terminal domain were lethal in vivo and inhibited Mcm2-7 loading onto origin DNA in vitro. I used the tagged proteins coupled with crosslinking and denaturing immunoaffinity purifications and found that Mcm3 interacts with Orc2 and Cdc6 during Mcm2-7/Cdt1 recruitment to ORC-Cdc6-DNA. The results of this thesis suggest that Mcm2-7 is recruited to origin DNA via Mcm3 interaction with Orc2 and Cdc6 and that the Mcm2-7 hexamers are loaded in a sequential manner.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available