CONTROL OF INITIATION OF DNA REPLICATION IN LUNG CANCER

这项提案的总体目标是识别DNA序列和 调节人体DNA复制启动的蛋白质因素 细胞。我们将利用这个实验室的两项新进展。 首先，我们将开发一种新的基于聚合酶链式反应的程序，允许快速 以及对大范围的起始点进行敏感的测绘 哺乳动物基因组DNA。肺肿瘤细胞系提供了一个独特的机会 因为不同的细胞系都有扩增的染色体 包含myc基因三个不同成员中的每一个的区域 一家人。我们现在已经定位了一个起始区 人类c-myc基因上游的复制。我们将继续 完善这一区域的地图，并确定初始 肺肿瘤细胞的双向复制。使用我们的方法，我们 应绘制N-myc和L-myc附近的复制起点图 基因。我们将确定复制的起源是否与 带有未扩增或扩增的myc家族基因的细胞。我们会 比较myc家族成员起始区的序列和 确定任何共识要素。其次，我们寻求迅速利用 关于我们最近发现的pur元件，一个16bp的共识序列 在几个复制的真核起源处保守。我们现在有了 确定了一种在主要DNA弯曲处与该元件结合的蛋白质 位于c-myc起始区。我们将提纯这种蛋白质，它 对富含嘌呤的PUR单链具有高度的、特异的亲和力。我们 将克隆这种蛋白质的基因并对其进行测序，这是我们正在进行的一个过程 已经成功开始。我们的目标是测试Pur结合蛋白， 以及任何其他潜在的启动因素，以提高 启动体外复制。我们将测试潜在的监管 特定双链开放活性和解旋酶活性的因素，两个 与启动过程开始相关的功能。 显示活性的蛋白质将在我们的完整体外测试中 选择最佳克隆序列的复制系统 效率。有趣的是，PUR存在于几个已知的基因附近 在各自的系统中放大。我们将对pur元素进行排序 近扩增的myc基因在肺癌细胞系中的表达 突变与扩增有关。结果将帮助我们 了解DNA复制如何启动以及此复制是如何进行的 在肺癌的特定起源上发生异常。

The overall goal of this proposal is to identify DNA sequences and protein factors regulating the initiation of DNA replication in human cells. We shall capitalize on two new developments from this laboratory. Firstly, we shall exploit a new PCR-based procedure that allows rapid and sensitive mapping of initiation sites over long stretches of mammalian genomic DNA. Lung tumor cell lines offer a unique opportunity for mapping since different cell lines possess amplified chromosome regions containing each of three different members of the myc gene family. We have at this time localized a zone of initiation of replication upstream of the human c-myc gene. We shall continue to refine mapping of this zone and to identify sites of initial bidirectional replication in lung tumor cells. Using our method, we shall map origins of replication in the vicinity of N-myc and L-myc genes. We shall determine whether origins of replication are the same in cells with either unamplified or amplified myc family genes. We shall compare sequences at initiation zones for the myc family members and identify any consensus elements. Secondly, we seek to quickly capitalize on our recent discovery of the PUR element, a 16 bp consensus sequence conserved at several eukaryotic origins of replication. We have now identified a protein that binds to this element at the major DNA bending site in the c-myc initiation zone. We shall purify this protein, which has high, specific affinity for the purine-rich single strand of PUR. We shall clone and sequence the gene for this protein, a process we have already successfully begun. Our aim is to test the PUR-binding protein, and any other potential initiation factor, for ability to promote initiation of replication in vitro. We shall test potential regulatory factors for specific duplex-opening activity and helicase activity, two functions associated with the beginning of the initiation process. Proteins exhibiting activity will be tested in our complete in vitro replication system with cloned sequences selected for optimal efficiency. Intriguingly, PUR is present near several genes known to be amplified in their respective systems. We shall sequence the PUR element near amplified myc genes in lung tumor cell lines to see whether mutations are associated with amplification. Results will help us understand how DNA replication initiates and how this replication is aberrant at specific origins in lung cancer.