JOINT STUDY ON FUNCTION OF RCC1 PROTEIN IN CELL CYCLE.

该项目分为两部分。首先是证明RCC1蛋白对DNA复制起始的要求。第二是表明RCC1蛋白在酶上起作用，而不是在结构上起作用。1) BHK细胞的RCC1基因温度敏感突变体不能维持正确的细胞周期时间顺序，在限制性温度下，染色体未完成S期就会过早凝结，进入有丝分裂。我们使用爪蟾卵提取物来研究RCC1在间期核功能中的作用，以及这种作用如何导致已知的温度敏感RCC1突变体的表型。通过从卵子提取物中提取免疫缺失的RCC1蛋白，我们发现它既不是染色质去浓缩所必需的，也不是核形成所必需的，但这对于添加的精子染色质DNA的复制是绝对必要的。我们的研究结果进一步表明，RCC1不以酶促的方式参与复制，但可能是形成或维持复合体所需的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，从而在DNA复制完成之前阻止有丝分裂的开始。2) 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，但不催化c-H-ras p21蛋白。在此基础上，我们首先确认RCC1在Ran上具有GEP活性，然后询问RCC1的GEP功能是否为DNA复制起始所必需。为此，我们根据丙氨酸扫描法构建了大量突变的RCC1蛋白。那些没有GEP活性的突变体没有启动精子DNA复制，但具有GEP功能的突变体启动了精子DNA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DNA复制的DNA起始所必需的。因此，RCC1酶作用于细胞而非结构。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