JOINT STUDY ON FUNCTION OF RCC1 PROTEIN IN CELL CYCLE.

该项目分为两个部分。首先是证明RCC 1蛋白是DNA复制起始的必需条件。1）BHK细胞RCC 1基因中的温度敏感突变体不能维持细胞周期的正确时间顺序，并且将过早地浓缩其染色体并在限制性温度下进入有丝分裂而没有完成S期。我们已经使用爪蟾卵提取物来研究RCC 1在间期核功能中的作用，以及这种作用如何有助于已知的温度敏感RCC 1突变体的表型。通过从卵提取物中免疫耗竭RCC 1蛋白，我们发现它既不是染色质去凝聚也不是核形成所需的，但绝对是复制添加的精子染色质DNA所需的。我们的研究结果进一步表明，RCC 1不参与复制酶，但可能是B ...更多信息 RCC 1是形成或维持复合物所需的结构复合物的一部分，RCC 1的缺失可能直接导致在DNA复制完成之前阻止有丝分裂起始的调节系统的破坏。2）据报道，RCC 1蛋白特异性催化Ran：ras相关的核蛋白，但不对蛋白c-H-rasp 21。在此基础上，我们首先证实了RCC 1对Ran具有GEP活性，然后探讨了RCC 1的GEP功能是否是DNA复制启动所必需的。为此，我们根据丙氨酸扫描法构建了大量突变的RCC 1蛋白。不具有GEP活性的突变体不能启动精子DNA的复制，而具有GEP功能的突变体则能启动精子DNA的复制。虽然我们的数据仍然是初步的，但这些数据表明，GEP功能是启动DNA复制所必需的。因此，RCC 1在细胞上以酶的方式而不是结构性地发挥作用。少

The project was divided into two parts. The first is to prove the requirement of RCC1 protein for initiation of DNA replication. The second is to indicate that the RCC1 protein functions enzymatically rather than structurally.1) Temperature-sensitive mutants in the RCC1 gene of BHK cells fail to maintain a correct temporal order of the cell cycle and will prematurely condense their chromosomes and enter mitosis at the restrictive temperature without having completed S phase. We have used xenopus egg extracts to investigate the role that RCC1 plays in interphase nuclear funtions and how this role might contribute to the known phenotype of temperature -sensitive RCC1 mutants. By immunodepleting RCC1 protein from egg extracts, we find that it is required for neither chromatin decondensation nor nuclear fornation but that is absolutely required for the replication of added sperm chromatin DNA. Our results further suggest that RCC1 does not participate enzynatically in replication but may b … More e part of a structural complex which is required for the formation or maintenance complex, the loss of RCC1 might lead directly to disruption of the regulatory system which prevents the initiation of mitosis before the completion of DNA replication.2) The RCC1 protein has been reported that it specifically catalyse the exchange of guanine nucleotides on the Ran : ras- related nuclear protein, but not on the protein c-H-ras p21. Based on this report we confirmed that the RCC1 has a GEP activity on the Ran at first, and then ask whether the GEP function of the RCC1 is required for the initiation of DNA replication. To do this, we construct a lot of mutated RCC1 proteins according to the alanine scanning method. Those mutant without the GEP activity did not initiate sperm DNA replication, but did the mutants with the GEP function. Although our data are still preliminary, those indicate that the GEP function is required for the initiation of DNA of DNA replication. Thus, the RCC1 enzymatically functions on the cell rather than structurally. Less