Tugal, Tamara; (2000) Characterisation of Xenopus Origin Recognition Complex. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The Origin Recognition Complex (ORC) of S. cerevisiae plays a crucial role in recognition of origins of DNA replication within the yeast genome. In Xenopus egg extracts DNA replication initiates from random DNA sequences suggesting that origins of DNA replication are not required. Despite this apparent lack of requirement for DNA sequences serving as origins of DNA replication, a protein homologous to the Ore Ip subunit of yeast ORC has been identified in Xenopus. This discovery has indicated that the mechanism which recognises replication origins in higher eukaryotes could be similar to that in yeast. Work described in this thesis started at the time when Xenopus Ore Ip was identified. In the quest to understand Xenopus ORC and it's function in origin recognition I set out to purify this protein complex. I produced monoclonal antibodies which recognised Xenopus Ore Ip subunit of ORC with high affinity and specificity. Using these antibodies, I purified a protein complex which contained Xenopus Ore Ip in association with at least four other polypeptides. I showed that one of these polypeptides was Xenopus Orc2p which was meanwhile identified by an independent research. Microsequencing of another Ore Ip associated protein led to identification of mouse Orc4 gene from the EST database. I used the identified mouse Orc4 sequence to screen Xenopus cDNA library and I cloned Xenopus Orc4 gene. Microsequencing of another two proteins co-purified with Ore Ip confirmed that they were Xenopus Orc3p and Orc5p and helped identification of their corresponding genes from frogs and humans. I described a mobility shift of Xenopus Ore Ip occurring upon the exit from mitosis and showed that it correlated with dephosphorylation of the protein. I determined that the amount of ORC present in a single frog egg is 100 000 fold higher than in a single somatic cell. Finally, I showed that ORC purified from Xenopus egg extract binds to S. cerevisiae ARSl sequence, as well as to DNA sequences derived from a bacterial plasmid. I observed that this ORC-DNA interaction was dependent on ATP. However, the way by which ORC determines the sites of initiation of DNA replication in higher eukaryotes remains unknown.

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