Developmental control of replication by Drosophila RB

果蝇 RB 对复制的发育控制

• 批准号: 6901134
• 负责人: GIOVANNI BOSCO
• 金额: $ 25.59万
• 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/6901134
• 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）：视网膜母细胞瘤肿瘤抑制蛋白（RB）是细胞增殖的关键调节因子。 RB 与 E2F 转录因子结合，共同抑制 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 还具有在复制起始位点调节 DNA 复制的直接功能。这种直接作用的证据来自五个观察结果：(1) RB 定位于原代人类细胞的复制灶，并调节 S 期以及 G1-S 转变的进展。 (2) RB与MCM7复制因子相互作用，可以抑制爪蟾无细胞和无转录系统中的复制。 (3) DNA损伤后RB可以被募集到哺乳动物复制起点并防止核内复制。 (4) 果蝇 RB (RBF) 与复制起始因子、起源识别复合体 (ORC) 相互作用。 (5) 果蝇E2F和RB中的突变体未能限制复制起始并使ORC在卵巢卵泡细胞中错误定位。 RB 直接控制复制的机制尚不清楚。该提议建立在我们之前的观察基础上，即果蝇 RB 与 ORC 相关，并且 RB 突变体使 ORC 定位错误并且未能限制 DNA复制。我们现在已经证明 RBF 的 N 末端（a.a. 151-330）是保守的 在整个 RB 家族蛋白中，对于其与 ORC 的相互作用至关重要。这个和最近关于人类 RB 定位于复制起始位点的报道强烈表明 RB 对复制起始的控制是进化上保守的功能。我们的中心假设是，RB 通过两种不同的机制调节 DNA 复制：首先，它通过响应抗增殖和发育线索来抑制 S 期特定基因的转录；其次，RB在起始点与ORC结合以抑制复制起始。我们在这里要问两个大问题。 (1) RB直接抑制复制起点的机制是什么？ (2) 内循环和绒毛膜扩增如何受发育信号调节，从而协调发育与 RB 调节的细胞周期事件？使用果蝇卵子发生作为模型系统，我们提出的研究将为我们提供关于 RB 肿瘤抑制蛋白如何响应发育线索调节 DNA 复制的新见解。