In vitro reconstitution of the replication initiation complex

复制起始复合物的体外重建

• 批准号: 13680757
• 负责人: SHIRAKATA Masaki
• 金额: $ 2.3万
• 依托单位: Tokyo Medical and Dental University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13680757/
• 关键词: Replication; EBV; Initiation complex; ORC

The aim of this research is to reconstitute the replication initiation complex on the latent replication origin of Epstein Barr virus oriP from synthesized polypeptides of the replication initiation factors. These replication factors include the cellular replication licensing factors MCMs, cdc6, and the viral replication factor EBNA1, which is the only virus-encoded polypeptide required for the replication from oriP We expressed these polypeptides in insect cells using Baculo virus vector and prepared substantial amount of MCMs and cdc6 However, it was difficult to synthesize the full length EBNA1 protein. This problem was solved at last by synthesizing an EBNA1 mutant that lacked the Gly-Ala repeat in the amino terminal domain Moreover, a transient replication assay confirmed that the mutant held the replicator activity of the wild type. It has taken almost a year to solve this problem, and thereby the reconstitution experiment is still preliminary In addition to the reconstitution experiment, we investigated regulation of the oriP replication further in detail, and found that oriP is negatively regulated by the signal transduction mediated by TRAF5 and TRAF6 through p38 MAPK We also identified Thr534 of EBNA1, which is important for the negative regulation by p38 MAPK. The EBNA1 mutants of which Thr534 is substituted with Glu may provide a useful tool to examine the specificity of oriP replication in the reconstituted system.

本研究的目的是从复制起始因子的合成多肽中重建EB病毒oriP潜伏复制起点上的复制起始复合物。这些复制因子包括细胞复制许可因子MCM、cdc6和病毒复制因子EBNA1，EBNA1是oriP复制所需的唯一病毒编码的多肽。我们使用杆状病毒载体在昆虫细胞中表达这些多肽，并制备了大量的MCM和cdc6。然而，合成全长EBNA1蛋白很困难。最终通过合成氨基末端结构域中缺少Gly-Ala重复的EBNA1突变体解决了这个问题。此外，瞬时复制测定证实该突变体保留了野生型的复制活性。解决这个问题花了将近一年的时间，因此重构实验还处于初步阶段。除了重构实验之外，我们还进一步详细研究了oriP复制的调控，发现oriP受到TRAF5和TRAF6通过p38 MAPK介导的信号转导的负调控。我们还鉴定了EBNA1的Thr534，这对p38 MAPK的负调控很重要。 Thr534 被 Glu 取代的 EBNA1 突变体可能提供有用的工具来检查重构系统中 oriP 复制的特异性。

项目成果

K Hirai, and M. Shirakata.: "Replication licensing of the EBV oriP minichromosome"Current Topics in Microbiology and Immunology. 258. 13-33 (2001)

K Hirai 和 M. Shirakata.：“EBV oriP 微型染色体的复制许可”微生物学和免疫学当前主题。

M Shirakata, K Imadome, K Okazaki, and K Hirai.: "Activation of TRAF5 and TRAF6 signal cascades negatively regulates the latent replication origin of Epstein-Barr virus through p38 mitogen-activated protein kinase"J. Virol.. 75. 5059-5068 (2001)

M Shirakata、K Imadome、K Okazaki 和 K Hirai.：“TRAF5 和 TRAF6 信号级联的激活通过 p38 丝裂原激活蛋白激酶负调节 Epstein-Barr 病毒的潜在复制起点”J。

白形正樹: "Novel immediate-early protein IE19 of human cytomegalovirus activates origin recognition complex I promoter in a coopeative manner with IE72"Journal of Virology. 76. 3158-3167 (2002)

Masaki Shirakata：“人巨细胞病毒的新型立即早期蛋白 IE19 以与 IE72 合作的方式激活起源识别复合物 I 启动子”病毒学杂志 76. 3158-3167 (2002)。

白形正樹: "Replication licensing of the EBV oriP minichromosome"Current Topics in Microbiology and Immunology. 258. 13-33 (2001)

Masaki Shirakata：“EBV oriP 微型染色体的复制许可”微生物学和免疫学当前主题 258. 13-33 (2001)。