Analysis for spatiotemporal regulation of DNA replication intiation proteins on human globin loci.

本项目的目的是了解哺乳动物细胞中DNA复制起始蛋白如ORC、CDC 6和MCM的时空调控。我们研究了这些蛋白质的核组织和细胞周期控制。我们发现ORC 1和ORC 2蛋白与核基质相关，并相互作用。因此，ORC 1可能与ORC 2、3、4和5在核基质上形成复合物，可能在DNA复制中起作用。ORC 1蛋白在S期通过泛素-蛋白酶体依赖途径降解。这可能有助于与核基质结合，尽管它们与ORC 1的物理缔合尚未检测到。另一方面，我们的研究结果表明，大多数MCM复合物更广泛地分布在染色质以外的ORC焦点。这种组织可以解释哺乳动物细胞中提出的“DNA复制起始区”。我们用染色质免疫沉淀法测定了这些蛋白质与人珠蛋白基因座染色体DNA的结合模式，该基因座被认为是DNA复制单位。我们首先建立了半定量PCR方法，检测了该基因座上的7个代表性片段，这些片段含有β-珠蛋白基因上游的假定复制起点。我们目前与异步HeLa细胞的初步结果表明，MCM复合物可能与所有地区的测试，与一些优先结合的起源。我们尚未获得关于CDC 6和ORC结合模式的指示性结果。我们继续尝试用染色质免疫沉淀法获得结论性的数据。

The purpose of this project is to understand spatiotemporal regulation of DNA replication initiation proteins such as ORC, CDC6 and MCM in mammalian cells. We have investigated nuclear organization and cell cycle control of these proteins. We found that ORC1 and ORC2 proteins are associated with the nuclear matrix and physically interact with each other. Therefore, it is likely that ORC1 forms a complex with ORC2, 3, 4 and 5 on the nuclear matrix, probably functioning in DNA replication. ORC1 proteins were found to be degraded during S phase by an ubiquitin-proteasome dependent pathway. This may contribute to bind to the nuclear matrix although their physical association with ORC 1 has been undetectable. On the other hand, our results suggest that most MCM complexes are more widely distributed on chromatin beyond ORC foci. Such organization could provide an explanation for "initiation zone of DNA replication" suggested in mammalian cells. By chromatin immunoprecipitation assay, we have tried to determine the binding pattern of these proteins to chromosomal DNA of human globin locus, which has been suggested to act as a DNA replication unit. We first established semi-quantitative PCR assay to detect seven representative fragments on the locus, containing a putative replication origin upstream of beta-globin gene. Our current preliminary results with asynchronous HeLa cells suggest that MCM complexes may be associated with all regions tested, with some preferential binding to the origin. We do not yet obtain indicative results regarding CDC6 and ORC binding pattern. We continue trying to obtain the conclusive data with chromatin immunoprecipitation assay.