Keeley, Anthony John; (1999) Holliday junction processing enzymes in eukaryotes. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Homologous genetic recombination is best understood in the model prokaryote Escherichia coli where the enzymatic processes have been characterised using purified proteins. Recombination involves reciprocal exchange of strands between two homologous DNA strands. The resultant Holliday junction is either branch migrated and/or resolved. Proteins, RuvA and RuvB can branch migrate the junction and RuvC can resolve the junction. In contrast limited advancement has been made with eukaryotic systems. A study was undertaken to elucidate some of the processes that occur in eukaryotic recombination. Activities capable of processing Holliday junction-containing substrates were screened for. In addition activities capable of processing Holliday junction precursor substrates, were also investigated. Initial searches involved assaying fractionated eukaryotic cell extracts for processing activity. A later approach was to PCR potential eukaryotic homologues of prokaryotic proteins. Both approaches were successful. The latter approach resulted in the identification of the homologue of CCE1 in S. cerevisiae being identified in S. pombe. The open reading frame of YDC2_SCHPO (spCCEl) was cloned, purified, assayed biochemically and found to be a Holliday junction specific resolvase. Initial characterisation of the protein was carried out. YDC2 being a homologue of CCE1 meant that it was likely to be a mitochondrial Holliday junction resolvase activity that could be masking a nuclear Holliday junction resolving activity. To search for another resolvase activity, a possible nuclear activity, a S. cerevisiae yeast strain that was a CCE1 knock out was used to resume the search for such possible activities. An initial fractionation of the knockout strain of S. cerevisiae revealed the presence of another Holliday junction resolving activity. This activity was partially purified and characterised. Similar searches in mammalian cells led to the identification of an end joining activity that in experiments seemed to show a homology dependency and an annealing activity.

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