HUMAN C MYC GENE REPLICATION ORIGIN

DNA复制过程是用于治疗癌症的抗增殖药物的主要生理靶点。我们工作的主要目标是描述哺乳动物DNA复制中主要的细胞周期调节步骤，即激活起源以启动DNA合成。我们的实验集中在人类c-myc复制的起源。了解DNA元件在c-myc起源中的功能，可能会为通过控制正常和疾病状态下细胞分裂的机制调节DNA代谢提供新的见解。c-myc起源是在后生动物中发现的有限数量的染色体起源之一，也是唯一在转染细胞和体外染色体起始位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S. cerevisae 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。该系统是高效的，可重复地将c-myc起源结构精确地定位到特定的基因组受体位点。FLP重组酶系统在构建的范围内是非常灵活的，可以在体内染色体环境中进行测试。因此，该系统不仅局限于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，我们将验证c-myc起源损害DNA合成首选起始位点的假设，并且这些位点的起始依赖于顺式作用复制子元件。在三个特定目标中，将在确定的染色体受体位点上整合一组c-myc起源构建体，并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。目的1将创造性地渐进的5‘或3’缺失，以及特定候选复制子元件的突变，用于分析起源活性。目的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位或端粒位置效应的影响。目的3将测试复制时间是否受到活性转录单元或端粒位置的影响。目的3将测试在c-myc起源中是否存在多个优先的起始位点，这些起始位点服从于顺式作用复制子。起源活性和结构将通过竞争性PCR、新生DNA链PCR定位、DNA酶消化、化学足迹和连接介导PCR来分析。

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.