CANCER SUSCEPTIBILITY AND S PHASE INITIATION SITES

哺乳动物细胞在暴露时最容易致癌 在同步化过程中，在S期的一开始， 增殖申请人假设，这种脆弱性可能是 与致癌物诱导的DNA调节区域的改变有关 控制S期开始的复制起点。因此 申请人试图找到在S早期激活的复制起点， 相位尽管最近在理解DNA复制是如何 起源于原核生物，病毒和酵母，很少有人知道DNA是如何 复制在S期开始时启动， 起源在哺乳动物细胞中以受调节的顺序方式被激活。 关于什么构成复制起点的知识非常有限， 高等真核生物人类细胞的复杂基因组可能利用不同的 复制的类型起源于一个协调和渐进的过程， S期复制。当细胞进入S期时被激活的起源 可能属于不同的类，而不是那些稍后激活的类， 基因组的有序复制该项目提出了一项战略，开始， 解决这个问题。申请人构建了DNA的粘粒文库， 作为正常人成纤维细胞（NHF 1）复制的序列进入S期。 他们将在这些文库中搜索具有高窝藏潜力的克隆体， 复制的起源早期复制DNA与核基质相关DNA 将用于鉴定与两种类型的探针杂交良好的克隆 (Aim 1）。克隆的人类DNA插入片段的末端将被测序， 用于检索保存在DNA数据库中的较长序列的信息。的 序列将被分析用于预测基因的结构基序的聚类。 潜在的复制起点功能（目标2）。为了证实 这些序列作为复制起点，功能性测定将是 基于竞争性PCR来测量它们在 来自增殖的人成纤维细胞的小的新生DNA（Aim 3）。候选 克隆也将用于确定其它细胞类型（淋巴母细胞样 和上皮细胞）在S期开始时共享相同的复制起点 (Aim 4）。最后，申请人希望确定 位于或接近初始复制起点的序列是 癌细胞这种病变可以解释他们观察到的易受 早期S期的致癌作用。调控序列中的突变， 在S期开始时控制复制的起始可能代表 放松细胞增殖调节的机制。这些 改变可能是另一种类型的遗传变化， 致癌作用

Mammalian cells are most vulnerable to carcinogenesis when exposed to DNA damaging agents at the very beginning of the S phase during synchronized proliferation. The applicants hypothesize that this vulnerability may be associated with carcinogen-induced alterations of regions of DNA that regulate the origins of replication that control the start of the S phase. Thus applicants seek to find origins of replication that are activated in early S phase. Despite recent advances in understanding how DNA replication is initiated in prokaryotes, viruses and yeast, little is known about how DNA replication is initiated at the start of S phase and how multiple subsequent origins are activated in a regulated, sequential manner in mammalian cells. There is very limited knowledge of what constitutes an origin of replication in higher eukaryotes. The complex genome of human cells may utilize different types of replication origins in a coordinated and progressive process of replication during S phase. Origins that are activated as cells enter S phase might belong to a different class than those that activate later, resulting in orderly duplication of the genome. This project proposes a strategy to begin to address this issue. The applicants constructed a cosmid library of DNA sequences that are replicated as normal human fibroblasts (NHF1) enter S phase. They will search these libraries for clones with high potential of harboring origins of replication. Early replicating DNA and nuclear matrix-associated DNA will be used to identify clones that hybridize well with both types of probes (Aim 1). The ends of the cloned human DNA inserts will be sequenced and this information used to retrieve longer sequences deposited in DNA databases. The sequences will be analyzed for clustering of structural motifs that predict the potential for replication origin function (Aim 2). To confirm the potential of these sequences to serve as replication origins, a functional assay will be used based on competitive PCR to measure their increased relative abundance in small nascent DNA from proliferating human fibroblasts (Aim 3). Candidate clones also will be used to determine whether other cell types (lymphoblastoid and epithelial cells) share the same origins of replication at the start of S (Aim 4). Ultimately the applicants wish to determine whether changes in sequences at or near the initial origins of replication are a common feature of cancer cells. Such lesions could explain their observation of vulnerability to carcinogenesis in the early S phase. Mutations in regulatory sequences that control the initiation of replication at the start of the S phase may represent a mechanism by which the regulation of cell proliferation is relaxed. These alterations may be another type of genetic change that initiates the process of carcinogenesis.