Developmental control of replication by Drosophila RB

果蝇 RB 对复制的发育控制

• 批准号: 7070511
• 负责人: GIOVANNI BOSCO
• 金额: $ 25.07万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7070511
• 关键词: Baculoviridae; DNA replication; Drosophilidae; arthropod genetics; cell growth regulation; cell proliferation; chorioallantoic membrane; chromatin immunoprecipitation; cytogenetics; gene induction /repression; genetic regulation; oogenesis; protein protein interaction; protein structure function; retinoblastoma protein; site directed mutagenesis; tissue /cell culture; transfection

DESCRIPTION (provided by applicant): The retinoblastoma tumor suppressor protein (RB) is a critical regulator of cell proliferation. RB binds to the E2F transcription factor and together repress transcription of S-phase genes, thereby controlling the G1-S cell cycle transition. It has become increasingly clear that RB also has a direct function in regulating DNA replication at sites of replication initiation. The evidence for this direct role comes from five observations: (1) RB localizes to replication foci in primary human cells and regulates progression through S-phase as well as the G1-S transition. (2) RB interacts with the MCM7 replication factor and can repress replication in a Xenopus cell-free and transcription-free system. (3) RB can be recruited to mammalian replication origins after DNA damage and prevents endoreduplication. (4) Drosophila RB (RBF) interacts with the replication initiation factor, the Origin Recognition Complex (ORC). (5) Mutants in Drosophila E2F and RB fail to limit replication initiation and mis-localize ORC in ovarian follicle cells. The mechanisms by which RB functions to control replication directly are not known. This proposal builds on our previous observations that Drosophila RB associates with ORC and that RB mutants mislocalize ORC and fail to limit DNA replication. We now have shown that the N-terminus of RBF (a.a. 151-330), that is conserved throughout RB family proteins, is critical for its interaction with ORC. This and recent reports that human RB localizes to replication initiation sites strongly suggests that RB control of replication initiation is an evolutionarily conserved function. Our central hypothesis is that RB regulates DNA replication by two distinct mechanisms: First, it represses transcription of S-phase specific genes by responding to antiproliferative and developmental cues; Second, RB associates with ORC at origins to inhibit replication initiation. There are two big picture questions we are asking here. (1) What are the mechanisms by which RB directly represses replication initiation at origins? (2) How are endo cycles and chorion amplification regulated by developmental signals thus coordinating development with RB regulated cell cycle events? Using Drosophila oogenesis as a model system, our proposed studies will give us new insights as to how the RB tumor suppressor protein in response to developmental cues regulates DNA replication.

描述（由申请人提供）：视网膜母细胞瘤抑制蛋白（RB）是细胞增殖的关键调节剂。 RB与E2F转录因子结合并加在一起抑制S期基因的转录，从而控制G1-S细胞周期转变。越来越清楚的是，RB在调节复制启动部位的DNA复制方面也具有直接功能。这种直接作用的证据来自五个观察结果：（1）RB本地将重复焦点定位为原代人细胞中的复制灶，并通过S期和G1-S转变调节进展。 （2）RB与MCM7复制因子相互作用，并可以在无爪蟾细胞和无转录系统中抑制复制。 （3）RB可以在DNA损伤后募集到哺乳动物复制起源，并防止内置。 （4）果蝇RB（RBF）与复制起始因子，原点识别复合物（ORC）相互作用。 （5）果蝇E2F和RB中的突变体无法限制卵巢卵泡细胞中的复制启动和错误定位的ORC。 RB直接控制复制的机制尚不清楚。该提案是基于我们以前的观察结果，即果蝇RB与ORC相关，RB突变体错误地定位了ORC，并且无法限制 DNA复制。我们现在已经表明，RBF的N末端（A.A. 151-330）是保守的 在整个RB家族蛋白中，对其与ORC的相互作用至关重要。这一和最近的报道说，人RB本地化到复制起始位点强烈表明，RB对复制起始的控制是进化保守的功能。我们的核心假设是，RB通过两种不同的机制调节DNA复制：首先，它通过响应抗增殖性和发育提示来抑制S期特异性基因的转录；其次，RB与ORC的起源相关联，以抑制复制启动。我们在这里问两个大问题。 （1）RB在起源处的复制启动的机制是什么？ （2）如何通过发育信号调节Endo循环和绒毛膜扩增，从而与RB调节的细胞周期事件协调发育？我们提出的研究将果蝇卵子发生作为模型系统，将为我们提供有关RB肿瘤抑制蛋白如何响应发育线索调节DNA复制的新见解。