HUMAN C MYC GENE REPLICATION ORIGIN

DNA复制过程是抗增殖药物治疗癌症的主要生理靶点。我们工作的广泛目标是表征哺乳动物DNA复制中主要的细胞周期调控步骤，即启动DNA合成的起始点的激活。我们的实验重点是人类c-myc的复制起点。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的细胞分裂的机制来调节DNA新陈代谢提供新的见解。C-myc起源是在后生动物中发现的为数不多的染色体起源之一，也是唯一在转基因细胞和体外染色体起始点的质粒中自主复制的起源。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，用于在人类细胞中定点整合DNA。这个系统是高效的，并且可以精确地将c-myc起始结构靶向到特定的基因组受体位置。FLP重组酶系统在可在体内染色体环境中测试的构建范围内非常灵活。因此，该系统不仅限于DNA复制的分析，而且广泛适用于DNA代谢的其他方面的研究。使用FLP系统，我们将检验这样的假设，即c-myc起源妥协了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的复制子元件。在三个特定目标中的每一个中，一组c-myc起源构建体将被整合在确定的染色体受体位置，并将评估野生型和突变起源整合前后这些位点的结构和复制活性。目标1将产生起始点5‘或3’的渐进性缺失，以及特定候选复制子元件的突变，用于分析起始点的活性。Aim 2将直接测试c-myc的起始活性或复制时间是否受到激活的转录单位或端粒位置效应的影响。AIM 3将测试复制时间是否受到激活的转录单位或端粒位置的影响。AIM 3将测试在c-myc起源中是否存在多个服从顺式作用复制子的启动DNA合成的首选起点。将通过竞争性聚合酶链式反应、新生DNA链的聚合酶链式反应图谱、DNA酶消化、化学足迹和连接介导的聚合酶链式反应来分析起始物的活性和结构。

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.