REPLICON MISFUNCTIONS IN HUMAN CARCINOGENESIS

人类细胞的恶性转化涉及多步骤的过程， 进行性遗传改变 虽然研究进展一直 在定义的结构和表达的变化突出 我们对DNA水平上发生的变化的了解 复制仍然非常差。 这项研究计划的目标是 来评估癌症相关复制错误的独特细节 in the human人beings生物. 癌细胞具有不受控制和不确定的 复制能力，表明在人类肿瘤细胞中的复制 与正常细胞中的复制有显著不同。 复制 人类基因组的启动涉及一个高度有组织的过程， 个体复制子，其可以是体细胞上或遗传上被破坏的 在致癌过程中。 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子 核糖体RNA（rRNA）基因座。 这个提议的力量和独创性 通过光催化DNA对人类基因组进行修饰 交联反应。 首先，因为这个过程可能取决于个人的敏感性， 复制子的化学致癌性损害，建议调查 DNA交联对rRNA复制子复制的影响。 具体来说，我们将描述的起始和终止网站的 通过分析DNA复制中的瞬时抑制， 并通过识别PUVA阻断的复制结构。 我们将 补充这一拓扑映射研究附加体复制 携带rRNA起始区和终止区的质粒DNA 未处理和PUVA处理的细胞中的基因座。 第二，因为稳定， 特定复制子激活中的体细胞获得性改变， 涉及人类细胞的恶性转化，建议 鉴定rRNA复制子复制时间的改变， 人类肿瘤细胞。 具体来说，我们将分析起始和延伸 在未处理和PUVA处理的细胞的S期， 细胞 我们将比较rRNA复制的时间模式 在正常和肿瘤细胞的S期， 染色体倍性。 第三，由于遗传获得的DNA改变 复制与癌症倾向有关，建议分析 来自癌症易感细胞的细胞中rRNA复制子的瞬时破坏 人类着色性干皮病变异综合征。 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 在rRNA复制子的不同功能区域中的PUVA损伤， 包括起始和终止位点。 这项拟议研究的长期目标是， 核糖体RNA基因座中的复制错误功能可能被视为 理解其他关键人类复制子中的改变的范例， 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.