REVISED ABSTRACT Replication initiation in eukaryotes is believed to be dependent on a six subunit, ATP-dependent complex of proteins, the Origin Recognition Complex (ORC), which loads the helicase MCM2-7 at origins of replication. In the last cycle of this grant we made the surprising discovery that several human cancer cell lines continue to proliferate and replicate their DNA in the absence of two important subunits of ORC, ORC1 or ORC2. This proposal will test whether cancer cell-lines survive through the action of a crippled ORC (ORC missing one subunit), or because cell transformation activates an alternate helicase-loading mechanism that allows MCM2-7 loading in the absence of the six subunit ORC. The proposal will also identify how human ORC activates and represses the compactness of the chromatin and thus regulate gene expression. It will test whether the individual subunits of ORC have functions in this regard only as the complex ORC or as individual proteins independent of the holo-ORC. The results will delineate the importance of ORC in replication initiation and maintenance of genome stability in cancer cells, identify ORC-bypass mechanisms and identify replication-independent functions of ORC in regulating cell physiology.
and Significance Regulation of genome stability is the hallmark of normal cells, and often deregulated in cancer cells. This project will determine whether the Origin Recognition Complex (ORC), till now believed to be essential for replication initiation, can be bypassed in cancer cells and the mechanism of this bypass. This will be very important for understanding how genome instability occurs in cancers. Non-homologous end-joining (NHEJ) mediated repair of double-strand breaks in DNA is important for the resistance of a cancer cell to radiotherapy and common types of chemotherapy. By implicating a novel family of protein kinases in the signal transduction from DSB to activation of NHEJ, the project will identify new targets for drugs that will increase the sensitivity of cancer cells to radiotherapy or chemotherapy.
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