Developmental control of replication by Drosophila RB

描述(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细胞周期的转变。越来越清楚的是，Rb在调节复制起始点的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：(1)Rb定位于原代人类细胞的复制中心，并通过S期和G1-S转换来调节进程。(2)Rb与MCM7复制因子相互作用，在非洲爪哇无细胞、无转录系统中抑制复制。(3)Rb可在DNA损伤后被募集到哺乳动物的复制起始点，并阻止内复制。(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的突变体不能限制ORC在卵泡细胞中的复制起始和错误定位。RB直接控制复制的机制尚不清楚。这一建议建立在我们之前观察到的果蝇Rb与ORC相关以及Rb突变体错位ORC并未能限制ORC的基础上 DNA复制。我们现在已经证明了RBF的N-末端(A.A.151-330)，这是保守的 在整个RB家族蛋白质中，是与ORC相互作用的关键。这一点以及最近关于人类RB定位于复制起始位置的报道强烈表明，RB控制复制起始是一种进化上保守的功能。我们的中心假设是，Rb通过两种不同的机制来调节DNA的复制：第一，它通过对抗增殖和发育信号的反应来抑制S期特定基因的转录；第二，Rb在起源上与ORC结合来抑制复制起始。我们在这里提出了两个大问题。(1)Rb直接抑制起始复制的机制是什么？(2)发育信号如何调节内切周期和绒毛膜扩增，从而协调发育与Rb调节的细胞周期事件？以果蝇卵子发生为模型系统，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提示调节DNA复制的新见解。

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.