Studies on ORC and double strand DNA break repair

修订后的摘要 真核生物的复制起始被认为依赖于一个六亚基的、依赖于ATP的蛋白质复合体，即起源识别复合体(Origin Recognition Complex，ORC)，它在复制的起始处加载解旋酶MCM2-7。在这笔赠款的最后一个周期中，我们有了一个令人惊讶的发现，几个人类癌细胞株在缺少ORC的两个重要亚单位ORC1或ORC2的情况下继续增殖和复制它们的DNA。这项提议将测试癌细胞系是通过残缺的ORC(ORC缺失一个亚基)的作用存活下来，还是因为细胞转化激活了一种替代的解旋酶加载机制，允许在缺少六个亚基的ORC的情况下加载MCM2-7。该提案还将确定人类ORC如何激活和抑制染色质的致密性，从而调节基因表达。它将测试ORC的各个亚基是否只作为复杂的ORC或作为独立于全息ORC的单个蛋白质具有这方面的功能。这些结果将阐明ORC在癌细胞复制启动和维持基因组稳定性中的重要性，识别ORC旁路机制，并识别ORC在调节细胞生理中的复制无关功能。

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.