Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.596432
Title: Functional analysis of Sulfolobus solfataricus MCM
Author: Barry, E. R.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2009
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Abstract:
The minichromosome maintenance (MCM) complex is thought to function as the replicative helicase in eukaryotes and archaea. It is a member of the AAA+ (ATPases associated with various cellular activities) protein family. In this thesis, I demonstrated that the isolated AAA+ domain of Sulfolobus solfataricus MCM is a functional helicase, however its catalytic properties are very different to those of the full length protein. The N-terminus of the protein is required to confer substrate specificity on the enzyme, and also to enhance processive helicase activity. Communication between DNA binding sites in the N- and C-termini of the protein is shown to depend on a highly conserved loop, which protrudes from the N-terminus of the protein towards the AAA+ domain, where it interacts with a DNA binding β-hairpin in a dynamic, nucleotide dependent manner. This communication is mediated between, rather than within, allowing a model to be proposed for how helicase activity is co-ordinated; both between DNA binding sites within subunits, but also between subunits around the ring. Finally, I investigated reports of phosphorylated forms of MCM in vivo and identify a phosphorylated serine residue in the AAA+ domains of endogenous S. solfataricus MCM. Mutant forms of the protein with phosphomimetics at this site have elevated helicase activity in vitro. To identify the kinase responsible, several S. solfataricus kinases are investigated biochemically, and one is identified which has high levels of kinase activity on MCM. This is shown to interact with MCM in vivo and to be regulated in a cell cycle dependent manner.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.596432  DOI: Not available
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