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、CDC6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ORC1和ORC2蛋白与核基质相关，并在物理上相互作用。因此，ORC1很可能与核基质上的ORC2、3、4和5形成一个复合体，可能在DNA复制中发挥作用。发现Orc1蛋白在S期通过泛素-蛋白酶体依赖的途径降解。这可能有助于与核基质结合，尽管它们与ORC 1的物理联系一直无法检测到。另一方面，我们的结果表明，大多数MCM复合体更广泛地分布在ORC焦点以外的染色质上。这样的组织可以为哺乳动物细胞中提出的“DNA复制起始区”提供解释。通过染色质免疫沉淀法，我们试图确定这些蛋白与人类珠蛋白基因座染色体DNA的结合模式，这被认为是一个DNA复制单位。我们首先建立了半定量PCR方法来检测该基因座上的七个具有代表性的片段，这些片段包含一个假定的复制来源在β-珠蛋白基因的上游。我们目前对异步HeLa细胞的初步结果表明，MCM复合体可能与所有被测试的区域相关，并与起源有一些优先结合。我们还没有得到关于CDC6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确凿的数据。

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.