Halford, Katie Anne; (2003) Biochemical Analysis Of Yeast Pre-Replicative Complex Assembly. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The boundaries of replication research would be greatly extended by the establishment of an origin-dependent in vitro DNA replication system. Saccharomyces cerevisiae is a prime candidate from which to develop such a system, as it initiates replication from specific, well conserved, short origin sequences. The first step towards producing a yeast cell free replication system was the development of a method to assemble pre-replicative complexes (pre-RCs) onto short repeat sequences of the origin ARS1, immobilised on Dynabeads. I have extended this research to use plasmids with ARS1 origins. The plasmids are competent for pre-RC assembly (ORC, Cdc6p, MCM) and this is specific for origin sequences with an A element, the element known to be essential for replication in vivo. Formation of the complexes is also time dependent and once the MCM complex has been loaded, it can stay associated even in the absence of the MCM loader Cdc6p. This is in accordance with in vivo data and suggests that the system will be useful to elucidate in vivo mechanisms of replication. I have also explored the kinetics of pre-RC assembly using different nucleotides and competitor ARS1 DNA. Different steps in pre-RC assembly have different nucleotide requirements and the loading of substantial levels of the MCM complex requires the hydrolysis of ATP. Loaded MCM complexes are stable on DNA, even in the presence of competitor ARS1 plasmid. I have further investigated the nucleotide requirements using mutants of Cdc6p with changes in an ATP interaction region. Recombinant Cdc6p was produced in E. coli and purified by means of a poly-histidine tag. Cdc6p or mutant cdc6p was also expressed as a second copy in yeast cells and extracts were produced containing these proteins. Experiments with rCdc6p or the yeast extracts produced comparable results. I have successfully established in vitro loading of pre-replicative complexes at ARS1 origins on plasmid DNA and begun to investigate the requirements of this system.

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