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靶标。我们工作的广泛目标是表征哺乳动物DNA复制的主要细胞周期调节步骤，即起源以启动DNA合成的激活。我们的实验侧重于人类C-MYC复制起源。了解DNA元素在C-MYC起源中的功能可能会通过控制正常和疾病状态的细胞分裂的机制来对DNA代谢的调节进行新的见解。 C-MYC起源是在后生动物中鉴定出的染色体起源数量有限，也是唯一在转染细胞中自主复制和在染色体起始位点体外自主复制的起源。为了测试突变对完整细胞染色体中C-MYC起源活性的影响，我们开发了一种创新的系统，基于酿酒酵母FLP重组酶，用于人类细胞中DNA的位点特异性整合。该系统高效，可重复地针对特定基因组受体位点的C-MYC起源构建体。 FLP重组酶系统在可以在体内染色体环境中测试的构建体中非常灵活。因此，该系统不限于对DNA复制的分析，而是广泛适用于对DNA代谢其他方面的研究。使用FLP系统，我们将检验以下假设：C-MYC来源会损害DNA合成的首选起始位点，并且在这些位点的起始取决于顺式作用复制器元素。在三个特定目的中的每一个中，C-MYC起源构建体将集成在定义的染色体受体位点，并在整合野生型和突变起源之前和之后的那些位点的结构和复制活动。 AIM 1将对来源的创造性进步5'或3'删除，以及特定候选复制器元素中的突变，以分析起源活动。 AIM 2将直接测试C-MYC来源活动或复制时机是否受主动转录单元或端粒位置效应的影响。 AIM 3将测试复制时间是否受主动转录单元或端粒位置影响的影响。 AIM 3将测试是否有多个首选起始位点用于在C-Myc来源中启动DNA合成，而这些启动位于顺式演员复制器中。起源活性和结构将通过竞争性PCR，新生DNA链的PCR映射，DNase消化，化学足迹和结扎介导的PCR进行分析。