JOINT STUDY ON FUNCTION OF RCC1 PROTEIN IN CELL CYCLE.

该项目分为两部分。一是证明RCC1蛋白是启动DNA复制的必要条件。1)BHK细胞RCC1基因的温度敏感型突变体未能维持细胞周期的正确时间顺序，并会在限制性温度下过早凝集染色体并进入有丝分裂，而不是完成S期。我们使用非洲爪哇的卵提取液来研究RCC1在间期核功能中所起的作用，以及这种作用可能如何影响已知的温度敏感型RCC1突变体的表型。通过对卵子提取物中RCC1蛋白的免疫去除，我们发现RCC1蛋白既不是染色质解缩所必需的，也不是核形成所必需的，而是复制添加的精子染色质DNA所必需的。我们的结果进一步表明，rcc1不参与复制，但可能是…RCC1是形成或维持复合体所需的更多结构复合体的一部分，RCC1的缺失可能直接导致调控系统的破坏，调控系统阻止在DNA复制完成之前启动有丝分裂。2)已有报道RCC1蛋白特异性催化RAN：RAS相关核蛋白上的鸟嘌呤核苷酸交换，而不是蛋白c-H-ras p21上的鸟嘌呤核苷酸交换。根据这一报道，我们首先证实了RCC1在RAN上具有GEP活性，然后询问RCC1的GEP功能是否是启动DNA复制所必需的。为此，我们根据丙氨酸扫描的方法构建了大量突变的RCC1蛋白。不具有GEP活性的突变株不启动精子DNA复制，而具有GEP功能的突变株则启动精子DNA复制。虽然我们的数据仍然是初步的，但这些都表明GEP功能是启动DNA复制所必需的。因此，RCC1在细胞上发挥酶的作用，而不是在结构上。较少

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