The Role of the DNA Unwinding Element Binding Protein, DUE-B, in DNA Replication

DNA复制的启动是细胞周期的关键控制点，因为 复制起始异常导致基因组不稳定、癌症等遗传性疾病 疾病。精确的起始控制需要复制前的有序组装 复制起始处的蛋白质和起始前蛋白质复合体。我们已经确定了一个 一种新的DNA解旋元件结合蛋白DUE-B，它特异性地与 本质上是由于人类c-myc的复制起源和其他起源。到期-B是 正常进入细胞周期的脱氧核糖核酸合成S阶段和 复制的启动。复制前复合体形成后的DUE-B功能 但在起始模板解开之前，并且需要加载预引发复合体 蛋白质CDC45和TopBP1位于起始处。因此，DUE-B与DUE-B非常相似 酵母CDC45负载蛋白SLD3，S期促进细胞周期蛋白的关键靶点。 依赖的蛋白激酶。 我们建议测试一个模型，其中后生动物DUE-B是 SLD3.增加了DUE B在复制中的作用的意义，我们发现了一个强大的 DUE-B过表达与卵巢癌的关系值得注意的是，DUE-B 也显示出第二种个性；蛋白质的N端75%的结构具有 在进化过程中高度保守，并显示氨基酰-tRNA校对 在酵母、细菌和古生菌中发现的活性。 我们将使用HeLa细胞和非洲爪哇卵萃取物来解决几个机制 DUE-B调节解旋酶激活剂结合的模型的一些方面 CDC45形成复制预起始复合体，其中Due-B依赖 Cdc45的装载是S相内DNA损伤检查点的一个目标。我们的专业 分析技术包括免疫印迹、染色质免疫沉淀、 定量聚合酶链式反应、荧光原位杂交和免疫荧光。在目标1中，我们将分析绑定 DUE-B和预起始复合蛋白对复制起始点的影响 S期内DNA损伤检查点对DUE-B功能的影响。目标2将描述 DUE-B靶向结构突变对其与蛋白质配体结合的影响 激活复制源。

The initiation of DNA replication is a critical control point of the cell cycle, since abnormal replication initiation leads to genome instability, cancer and other inherited diseases. Accurate control of initiation requires the ordered assembly of prereplication protein and preinitiation protein complexes at origins of replication. We have identified a novel DNA unwinding element binding protein, DUE-B, which binds specifically to the essential DUE of the human c-myc replication origin and other origins. DUE-B is necessary for normal entry into the DNA synthetic S phase of the cell cycle and for the initiation of replication. DUE-B functions after formation of the prereplication complex but before origin template unwinding, and is required to load the preinitiation complex proteins Cdc45 and TopBP1 at origins. DUE-B therefore shows strong similarity to the yeast Cdc45-loading protein Sld3, a key target of the S phase promoting cyclin- dependent kinases. We propose to test a model in which metazoan DUE-B is the functional homolog of Sld3. Adding significance to the role of DUE B in replication, we have found a strong correlation between DUE-B overexpression and ovarian cancer. Remarkably, DUE-B also shows a second personality; the structure of the N-terminal 75% of the protein has been strongly conserved during evolution, and displays aminoacyl-tRNA proofreading activities found in yeast, bacteria and archae. We will use HeLa cells and Xenopus egg extracts to address several mechanistic aspects of the model in which DUE-B regulates the binding of the helicase activator Cdc45 to form the replication preinitiation complex, and in which the DUE-B-dependent loading of Cdc45 is a target of the intra-S phase DNA damage checkpoint. Our major analytical techniques will be immunoblotting, chromatin immunoprecipitation, quantitative PCR, FRET, and immunofluorescence. In Aim 1 we will analyze the binding of DUE-B and preinitiation complex proteins to replication origins, and the effect of the intra-S phase DNA damage checkpoint on DUE-B function. Aim 2 will characterize the effects of targeted structural mutations in DUE-B on its binding to protein ligands and its activation of replication origins.