Studies on ORC and double strand DNA break repair

修订摘要 真核生物中的复制起始被认为依赖于六个亚基的ATP依赖性蛋白质复合物，即起始识别复合物（ORC），其在复制起点加载解旋酶MCM 2 -7。在这项资助的最后一个周期中，我们发现了一个令人惊讶的发现，几种人类癌细胞系在缺乏ORC的两个重要亚基ORC 1或ORC 2的情况下继续增殖和复制它们的DNA。该提案将测试癌细胞系是否通过受损ORC（缺失一个亚基的ORC）的作用存活，或者因为细胞转化激活了替代解旋酶加载机制，该机制允许在不存在六个亚基ORC的情况下加载MCM 2 -7。该提案还将确定人类ORC如何激活和抑制染色质的紧密性，从而调节基因表达。它将测试ORC的单个亚基在这方面是否仅作为复合ORC或作为独立于holo-ORC的单个蛋白质具有功能。这些结果将描述ORC在癌细胞复制起始和维持基因组稳定性中的重要性，确定ORC旁路机制，并确定ORC在调节细胞生理学中的复制独立功能。

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.