CANCER SUSCEPTIBILITY AND S PHASE INITIATION SITES

哺乳动物细胞暴露在环境中最容易致癌 对DNA损伤剂在S阶段的一开始进行同步 扩散。申请者假设该漏洞可能是 与致癌物诱导的DNA调控区域的改变有关 控制S阶段开始的复制起源。因此， 申请者寻求寻找S早期激活的复制起源 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，人们对DNA是如何 复制在S阶段开始时启动，以及多个后续 在哺乳动物细胞中，起源是以一种受调控的、顺序的方式激活的。 关于什么构成复制来源的知识非常有限 高等真核生物。人类细胞的复杂基因组可能利用不同的 复制的类型起源于协调和渐进的过程 S期复制。细胞进入S时被激活的起源 可能与稍后激活的类属于不同的类，从而导致 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。申请者构建了一个dna粘粒文库。 复制为正常人成纤维细胞的序列进入S期。 他们将在这些文库中搜索具有高隐蔽性的克隆 复制的起源。早期复制DNA与核基质相关DNA 将用于鉴定与这两种类型的探针杂交良好的克隆 (目标1)。克隆的人类DNA插入片段的末端将被测序，这 用于检索存放在DNA数据库中的较长序列的信息。这个 将对序列进行分析，以对预测 复制起始功能的潜力(目标2)。确认…的潜力 将这些序列作为复制起始点，进行功能分析 基于竞争性聚合酶链式反应来测量它们在 来自增殖的人成纤维细胞的小的新生DNA(目标3)。侯选人 克隆也将用于确定其他类型的细胞(淋巴母细胞 和上皮细胞)在S开始时有着相同的复制起源 (目标4)。归根结底，申请人希望确定是否更改了 在复制的初始起始处或附近的序列是 癌细胞。这样的损伤可以解释他们观察到的易感性 致癌时间为S早期。调节序列中的突变 在S阶段开始时控制复制的启动可能代表 放松对细胞增殖的调节的机制。这些 改变可能是另一种类型的基因改变，它启动了 致癌。

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.