REPLICON MISFUNCTIONS IN HUMAN CARCINOGENESIS

人类细胞的恶性转化涉及一个多步骤的过程 渐进性的基因改变。虽然研究进展一直是 在定义结构和表达上的变化方面表现突出 特定的基因，我们对DNA水平上发生的变化的了解 复制仍然极其糟糕。这项研究提案的目标是 评估与癌症相关的复制故障的独特细节 在人类身上。癌细胞具有不受控制和不确定的特性 复制能力，表明在人类肿瘤细胞中复制 与正常细胞中的复制有显著的不同。复制 人类基因组的研究涉及到一个高度有组织的激活过程 个体复制体，这可能是一种身体上的基因破坏 在癌变过程中。这里提出的研究将帮助我们测试某些 这一假设的关键方面，使用人类作为模型复制 核糖体RNA(RRNA)基因座。这项提议的力量和独创性 源于通过光催化的DNA对人类基因组的修饰 与补骨脂素的交联反应。 首先，因为这一过程可能取决于个人的敏感性 复制体对化学致癌的损害，建议进行研究 DNA交联对rRNA复制子复制的影响。 具体地说，我们将描述 通过分析DNA复制中的瞬时抑制来实现rRNA复制子 并通过鉴定被PUVA阻断的复制结构。我们会 通过研究的附体复制来补充这种拓扑图 携带rRNA起始区和终止区的质粒DNA 在未经处理和PUVA处理的细胞中。第二，因为稳定， 激活特定复制子的躯体获得性改变是 参与人类细胞的恶性转化，有人建议 确定rRNA复制子在复制时间上的变化 人类肿瘤细胞。具体地说，我们将分析启动和伸长 在未经处理和经PUVA处理的S期 细胞。我们将比较rRNA复制的时间模式 正常人和肿瘤细胞不同程度S期复制子的研究 染色体倍性。第三，由于基因获得的DNA改变 复制与癌症倾向有关，建议分析 癌症易感人群细胞中rRNA复制子的瞬时中断 人类色素性干皮病变异型。具体来说，我们将 确定XPV中的DNA复制是否对PUVA诱导的 链内单加合物。我们将研究XPV的复制缺陷 对于rRNA复制子不同功能区域中的PUVA损伤， 包括起始点和终止点。 拟议研究的长期目标是解开 核糖体RNA基因座的复制故障可能被视为 理解其他关键人类复制中的改变的范例， 并有望为癌症提供新的治疗方法。

Malignant transformation of human cells involves a multistep process of progressive genetic alterations. While research progress has been outstanding in defining alterations in the structure and expression of specific genes, our knowledge on changes occuring at the level of DNA replication is still extremely poor. The goal of this research proposal is to evaluate the unique details of cancer-related replication misfunctions in the human beings. Cancer cells possess uncontrolled and indefinite replication capacity, indicating that replication in human tumor cells differ in significant ways from replication in normal cells. Replication of the human genome involves a highly organized process of activation of individual replicons, which can be somatically an genetically disrupted during carcinogenesis. The studies proposed here will help us test certain critical aspects of this hypothesis, using as model replicon the human ribosomal RNA (rRNA) locus. The power and originality of this proposal derives from the modification of the human genome via a photocatalyzed DNA crosslinking reaction with psoralen. First, because the process may depend on the sensitivity of individual replicons to chemical carcinogenic damage, it is proposed to investigate the effect of DNA crosslinking on the replication of the rRNA replicon. Specifically, we will characterize the initiation and termination sites of the rRNA replicon by analyzing the transient inhibition in DNA replication and by identifying the replication structures blocked by PUVA. We will complement this topological mapping by studying the episomal replication of plasmid DNA carrying the initiation and termination regions of the rRNA locus in untreated and PUVA-treated cells. Second, because stable, somatically acquired alterations in the activation of specific replicons is involved in malignant transformation of human cells, it is proposed to identify alterations in the replication timing of the rRNA replicons in human tumor cells. Specifically, we will analyze initiation and elongation of the rRNA replicon throughout the S phase of untreated and PUVA-treated cells. We will compare the temporal pattern of replication of the rRNA replicon in the S phase of normal and tumor human cells with various degree of chromosomal ploidy. Third, since genetically acquired alteration of DNA replication are associated with cancer-proneness, it is proposed to analyze transient disruptions of the rRNA replicon in cells from the cancer-prone human syndrome xeroderma pigmentosum variant. Specifically, we will determine if DNA replication in XPV is uniquely sensitive the PUVA-induced intrastrand monoadducts. We will study the replication defect of XPV toward PUVA damage in distinct functional regions of the rRNA replicon, including the initiation and termination sites. The long-term aim of the proposed study is that the unraveling of replication misfunctions in the ribosomal RNA locus may be seen as a paradigm for understanding alteration in other critical human replicons, and hopefully may provide new therapies for cancer.