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HUMAN C MYC GENE REPLICATION ORIGIN

人类 C MYC 基因复制起源

基本信息

批准号：
6519707
负责人：
Michael LEFFAK
金额：
$ 29.08万
依托单位：
WRIGHT STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1996
资助国家：
美国
起止时间：
1996-03-01 至 2004-06-30
项目状态：
已结题

项目摘要

The process of DNA replication is the primary physiological target for anti-proliferative drugs used to treat cancer. The broad goal of our work is to characterize the major cell cycle regulated step in mammalian DNA replication, the activation of origins to initiate DNA synthesis. Our experiments focus on the human c-myc replication origin. Understand the function of DNA elements in the c-myc origin is likely to give new insight into the regulation of DNA metabolism by mechanisms that control cell division in normal and disease states. The c-myc origin is one of a limited number of chromosomal origins identified in metazoans, and the only origin to replicate autonomously in plasmids in transfected cells and in vitro at chromosomal initiation sites. To test the effects of mutations on c-myc origin activity in the chromosomes of intact cells, we have developed an innovative system based on the S. cerevisae FLP recombinase for the site-specific integration of DNA in human cells. This system is highly efficient, and reproducibly targets c-myc origin constructs with precision to specific genomic acceptor sites. The FLP recombinase system is extremely flexible in the range of constructs that can be tested in an in vivo chromosomal environment. Hence, the system is not limited to the analysis of DNA replication but is broadly applicable to the study of other aspects of DNA metabolism. Using the FLP system we will test the hypothesis that the c-myc origin compromises preferred start sites for DNA synthesis and that initiation at these sites depends on cis-acting replicator elements. In each of three Specific Aims a panel of c-myc origin constructs will be integrated at defined chromosomal acceptor sites and the structure and replication activity at those sites before and after integration of the wild type and mutated origins will be assessed. Aim 1 will creative progressive 5' or 3' deletions of the origin, and mutations in specific candidate replicator elements, for analysis of origin activity. Aim 2 will test directly whether c-myc origin activity or replication timing is affected by an active transcription unit or telomere position effects. Aim 3 will test whether replication timing is affected by an active transcription unit or telomere position affects. Aim 3 will test whether there are multiple preferred start sites for the initiation of DNA synthesis in the c-myc origin that are subservient to a cis-acting replicator. Origin activity and structure will be analyzed by competitive PCR, PCR mapping of nascent DNA strands, DNase digestion, chemical footprinting, and ligation-mediated PCR.

DNA复制过程是用于治疗癌症的抗增殖药物的主要生理目标。我们工作的总体目标是表征哺乳动物 DNA 复制中主要细胞周期调节步骤，即激活起点以启动 DNA 合成。我们的实验重点关注人类 c-myc 复制起点。了解 c-myc 起源中 DNA 元件的功能可能会为通过控制正常和疾病状态下细胞分裂的机制来调节 DNA 代谢提供新的见解。 c-myc起源是在后生动物中鉴定的有限数量的染色体起源之一，并且是在转染细胞中和体外在染色体起始位点的质粒中自主复制的唯一起源。为了测试突变对完整细胞染色体中 c-myc 起源活性的影响，我们开发了一种基于酿酒酵母 FLP 重组酶的创新系统，用于人类细胞中 DNA 的位点特异性整合。该系统非常高效，并且可重复地靶向 c-myc 起源构建体，精确地定位到特定的基因组受体位点。 FLP 重组酶系统的构建体范围极其灵活，可在体内染色体环境中进行测试。因此，该系统不仅限于DNA复制的分析，还广泛适用于DNA代谢其他方面的研究。使用 FLP 系统，我们将测试以下假设：c-myc 起源损害 DNA 合成的首选起始位点，并且这些位点的起始取决于顺式作用复制子元件。在三个具体目标中的每一个中，一组c-myc起源构建体将被整合到指定的染色体受体位点，并且将评估野生型和突变起源整合之前和之后这些位点的结构和复制活性。目标 1 将创造性地对起点进行 5' 或 3' 删除，并在特定候选复制子元件中进行突变，以分析起点活性。目标 2 将直接测试 c-myc 起源活性或复制时间是否受到活性转录单元或端粒位置效应的影响。目标 3 将测试复制时间是否受到活性转录单位或端粒位置的影响。目标 3 将测试 c-myc 起点中是否存在多个服从顺式作用复制子的 DNA 合成起始位点。将通过竞争性 PCR、新生 DNA 链的 PCR 作图、DNase 消化、化学足迹和连接介导的 PCR 来分析起始活性和结构。