Mapping Critical DNA Sequence Elements Required for Amplification of erbB2 in Breast Cancer
Annual rept. 3 Apr 2000-2 Apr 2003
BRIGHAM AND WOMEN'S HOSPITAL BOSTON MA
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To study the implications of replication initiator proteins to gene amplification, we characterized two proteins required for DNA replication ORC and Mcm10. In an attempt to purify recombinant human ORC from insect cells infected with baculoviruses expressing HsORC subunits, we found ORC2, -3, -4, and -5 forms a core complex. Expression of ORC3N, which inhibits ORC2-ORC3 interaction, causes cell cycle arrest. Consistent with this result, ORC2-depletion by RNAi arrested cell cycle in G1 with low CDK activity. We propose that cells have a novel mechanism to ensure that CDK2 is activated only after enough pre-RC is formed. We also report that chromatin-loading of Mcm10 requires a functional pre-RC but is independent of Cdc7 activity. Mcm10 is required for chromatin-binding of Cdc45 and origin unwinding. By using EBV replication system in human cells, we report that overexpression of geminin inhibits replication of episome, but not that of host cell chromosome, suggesting a novel means by which to cure cancer with gene amplification where the amplicons are carried as episomes. We also report that overexpression of Cdt1 and CDC6 results in rereplication in cells with nonfunctional p53, suggesting a novel function of p53 to prevent rereplication.
- Anatomy and Physiology
- Medicine and Medical Research