Analysis of the Human c-myc Gene Replication Origin

描述(由申请人提供)： DNA复制对细胞增殖至关重要，由于异常复制可能导致基因组不稳定、肿瘤形成或细胞凋亡，因此DNA复制受到严格监控。针对复制过程的药物是癌症化疗的核心，对复制控制的洞察可能会导致新的治疗策略。复制的主要细胞周期控制发生在复制子和复制起始点，其中DNA合成的启动依赖于多蛋白复合体的组装。 人类c-myc复制子将被用作模型系统。我们以前鉴定了c-myc复制子，并证明了它在内源性c-myc位点、在人类细胞中的转基因质粒、在体外复制的质粒中以及在HeLa基因组的异位位置上的活性。最近，我们使用酵母FLP重组酶将诱变的c-myc复制子结构定向到特定的基因组位置，并确定了对起始活性至关重要的序列。在酵母单杂交实验中，我们用其中一个序列作为诱饵，发现了c-myc DNA解卷元件结合蛋白DUE-B。我们将继续使用FLP重组酶系统来产生靶向整合c-myc复制子结构的克隆性人类细胞系。 我们将测试一个模型的几个方面，在该模型中，表观遗传因素指定复制起始位置，Due-B蛋白参与在假定的复制分叉加载DNA修复蛋白。目标1将通过甲基化或取代突变改变c-myc复制子的结构；目标2将评估蛋白质结合和复制子活性之间的关系。所有复制子突变体将在相同的染色体受体位置进行分析。复制子的活性和结构将通过定量PCR、DNA酶消化和染色质免疫沉淀进行分析。在目标3中，我们将使用物理、生化和免疫学方法来表征DUE-B蛋白及其在DNA复制中的作用。

DESCRIPTION (provided by applicant): DNA replication is essential for cell proliferation, and is under strict surveillance since abnormal replication can lead to genomic instability, tumor formation or apoptosis. Drugs that target the process of replication are central to cancer chemotherapy, and insight into the control of replication may lead to new treatment strategies. The primary cell cycle control of replication occurs at replicators and replication origins, where the initiation of DNA synthesis depends on the assembly of multiprotein complexes. The human c-myc replicator will be used as a model system. We previously identified the c-myc replicator and demonstrated its activity at the endogenous c-myc locus, in transfected plasmids in human cells, in plasmids replicated in vitro, and at an ectopic site in the HeLa genome. Recently, we used the yeast FLP recombinase to target mutagenized c-myc replicator constructs to a specific genomic site and identify sequences essential for origin activity. Using one of these sequences as bait in a yeast one-hybrid assay we discovered the c-myc DNA unwinding element binding protein DUE-B. We will continue to use the FLP recombinase system to generate clonal human cell lines with targeted integration of c-myc replicator constructs. We will test several aspects of a model in which epigenetic factors specify replication initiation sites and the DUE-B protein is involved in the loading of DNA repair proteins at presumptive replication forks. Aim 1 will alter the structure of the c-myc replicator by methylation or substitution mutagenesis; Aim 2 will assess the relationship between protein binding and replicator activity. All replicator mutants will be analyzed at the same chromosomal acceptor site. Replicator activity and structure will be analyzed by quantitative PCR, DNase digestion, and chromatin immunoprecipitation. In Aim 3 we will characterize the DUE-B protein and its role in DNA replication using physical, biochemical and immunological methods.