Developmental control of replication by Drosophila RB

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E2 F转录因子结合，共同抑制S期基因的转录，从而控制G1-S细胞周期转换。越来越清楚的是，RB在复制起始位点也具有调节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中的复制灶，并通过S期以及G1-S转换调节进展。(2)RB与MCM 7复制因子相互作用，可以抑制非洲爪蟾无细胞和无转录系统中的复制。(3)RB可以在DNA损伤后被募集到哺乳动物复制起点并阻止核内复制。(4)果蝇RB（RBF）与复制起始因子，起源识别复合物（ORC）相互作用。(5)果蝇E2 F和RB的突变体不能限制复制起始，并将ORC错误定位在卵泡细胞中。RB用于直接控制复制的机制尚不清楚。这一建议建立在我们以前的观察基础上，即果蝇RB与ORC相关，RB突变体错误定位ORC， DNA复制。我们现在已经表明，RBF（a.a. 151-330），这是保守的 在整个RB家族蛋白中，对于其与ORC的相互作用至关重要。这和最近的报告，人类RB定位于复制起始位点强烈表明，RB控制复制起始是一个进化保守的功能。我们的中心假设是RB通过两种不同的机制调节DNA复制：第一，它通过响应抗增殖和发育线索来抑制S期特异性基因的转录;第二，RB在起点与ORC相关联以抑制复制起始。我们在这里提出了两个大问题。(1)RB直接抑制复制起始的机制是什么？(2)发育信号是如何调控内膜周期和绒毛膜扩增的，从而协调发育与RB调控的细胞周期事件？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新的见解RB肿瘤抑制蛋白如何响应发育线索调节DNA复制。

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.