Analysis of the Human c-myc Gene Replication Origin

描述（由申请人提供）：DNA在复制过程中最容易受到损伤。因此，复杂的机制已经发展，以确保在复制起点的DNA合成的准确的空间和时间的启动，并在复制叉进展过程中的DNA的忠实复制。多蛋白复制起始复合物组分的错误，或肿瘤抑制蛋白无法解析分叉运动的阻滞，导致DNA重排、丢失或复制，并导致许多遗传疾病。我们的实验室确定了人类c-myc癌基因5'端的复制起点，并且我们已经表明，作为复制起点的核心c-myc复制子的结构、蛋白结合和功能在其内源性染色体位点和在人类基因组中的异位位点是相同的。在本申请中，c-myc复制子将被用作哺乳动物复制起点的模型，以研究使起点活性成为可能的DNA序列和蛋白质因子。此外，c-myc复制子将用于启动自然发生的疾病相关重复DNA序列的复制，这些序列是复制叉进展的障碍。参与检测和稳定停滞复制叉的蛋白质负责抑制基因组不稳定性、癌症和神经退行性疾病。由于哺乳动物染色质对复制施加了可能在其他模型系统中无法重现的限制，因此这是一种创新方法，可以表征影响染色质环境中复制叉稳定性的多个序列和DNA应激反应蛋白。所有三个目标都将使用定点FLP重组酶介导的盒式交换（FLP-RMCE）将c-myc复制子及其修饰的构建体整合到人类基因组中的一个独特位点。目的1将使用缺失分析通过定量染色质免疫沉淀（ChIP）来鉴定c-myc复制子中的蛋白结合位点，通过新生DNA的PCR定量（qPCR）来确定最小c-myc复制起点，并通过新生DNA的ChIP和qPCR来评估Gal 4将复制蛋白募集到起点的效果。目的2将测试在复制过程中的（CTG 7 CAG）n和（ATTCT 7AGAAT）n微卫星的扩展或收缩的起点位置，方向，重复序列组成的影响，和叉稳定蛋白的影响。在目标3中，我们已经证明形成天然停滞复制叉的来自人PKD 1/TSC 2基因座的天然存在的不对称聚嘌呤7聚嘧啶序列将从异位c-myc起点复制，并且复制体、DNA应激反应蛋白、移位酶和解旋酶在叉稳定和重启中的作用将通过新生DNA的siRNA敲低、ChIP和qPCR来评估。我们预计，这些研究将提供新的洞察过程中的复制起始，复制叉的进展，并在人类染色体环境中的基因组稳定。公共卫生相关性：DNA复制起始的不适当调节可导致染色体断裂、异常细胞分裂和人类疾病。我们已经开发出一种新的系统，模拟在癌症和神经退行性疾病中观察到的基因组不稳定性。因此，我们的研究很可能为DNA复制机制、遗传疾病的起源和疾病治疗的新模型提供新的见解。

DESCRIPTION (provided by applicant): DNA is most susceptible to damage during the process of replication. Therefore, complex mechanisms have evolved to ensure the accurate spatial and temporal initiation of DNA synthesis at replication origins, and the faithful copying of DNA during replication fork progression. Errors in the components of the multiprotein replication initiation complex, or the inability of tumor suppressor proteins to resolve blocks to fork movement lead to rearrangements, losses or duplications of DNA, and result in numerous genetic disorders. Our laboratory identified the replication origin 5' to the human c-myc oncogene, and we have shown that the structure, protein binding and function of the core c-myc replicator as a replication origin are the same at its endogenous chromosomal site and at ectopic sites in the human genome. In this application, the c-myc replicator will be used as a model mammalian replication origin to study the DNA sequences and protein factors that enable origin activity. In addition, the c-myc replicator will be used to initiate the replication of naturally occurring disease-related repeated DNA sequences that are impediments to replication fork progress. The proteins involved in sensing and stabilizing stalled replication forks are responsible for the suppression of genome instability, cancer, and neurodegenerative diseases. Because mammalian chromatin imposes constraints on replication that may not be recapitulated in other model systems, this is an innovative approach that allows the characterization of multiple sequences and DNA stress response proteins affecting replication fork stability in a chromatin environment. All three Aims will use site-directed FLP recombinase-mediated cassette exchange (FLP-RMCE) to integrate the c-myc replicator and its modified constructs into a unique site in the human genome. Aim 1 will use deletion analysis to identify protein binding sites in the c-myc replicator by quantitative chromatin immunoprecipitation (ChIP), define the minimal c-myc replication origin by PCR quantitation (qPCR) of nascent DNA, and assess the effect of Gal4 recruitment of replication proteins to the origin by ChIP and qPCR of nascent DNA. Aim 2 will test the effects of origin location, orientation, repeat sequence composition, and the effects of fork stabilizing proteins on the expansion or contraction of (CTG7CAG)n and (ATTCT7AGAAT)n microsatellites during replication. In Aim 3, a naturally occurring asymmetric polypurine7polypyrimidine sequence derived from the human PKD1/TSC2 locus, which we have shown to form a natural stalled replication fork, will be replicated from the ectopic c-myc origin and the role of replisome, DNA stress response proteins, translocase and helicases in fork stabilization and restart will be assessed by siRNA knockdown, ChIP, and qPCR of nascent DNA. We anticipate that these studies will give novel insight into the processes of replication initiation, replication fork progression, and genome stabilization in a human chromosomal environment. PUBLIC HEALTH RELEVANCE: Inappropriate regulation of DNA replication initiation can lead to chromosome fragmentation, abnormal cell division and human disease. We have developed a novel system that mimics the genomic instability observed in cancers and neurodegenerative diseases. Thus, our studies are likely to provide fundamental new insight into the mechanisms of DNA replication, the origins of genetic diseases, and new models for disease treatment.