Analysis of the Human c-myc Gene Replication Origin

描述(申请人提供)：DNA在复制过程中最容易受到损伤。因此，复杂的机制已经进化，以确保复制起始处DNA合成的准确空间和时间启动，以及复制分叉过程中DNA的忠实复制。多蛋白复制起始复合体成分的错误，或肿瘤抑制蛋白无法解决阻碍分叉运动的问题，会导致DNA重排、丢失或复制，并导致许多遗传疾病。我们的实验室已经确定了c-myc癌基因的复制起点5‘，并且我们已经证明了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染色体位置和在人类基因组的异位位置是相同的。在这一应用中，c-myc复制子将被用作一个模式哺乳动物复制起点，以研究使起点活性的DNA序列和蛋白质因子。此外，c-myc复制子将被用于启动自然发生的与疾病相关的重复DNA序列的复制，这些重复DNA序列是复制分叉进展的障碍。参与感知和稳定停滞复制叉的蛋白质负责抑制基因组不稳定、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限制，这在其他模型系统中可能无法概括，这是一种创新的方法，可以在染色质环境中表征影响复制分叉稳定性的多个序列和DNA应激反应蛋白。这三个AIMS都将使用定点定向FLP重组酶介导盒交换(FLP-RMCE)将c-myc复制子及其修饰的构建物整合到人类基因组中的一个独特位置。目的1通过定量染色质免疫沉淀法(ChIP)确定c-myc复制子中的蛋白结合部位，通过对新生DNA进行定量(QPCR)来确定c-myc的最小复制起点，并通过ChIP和新生DNA的qPCR来评估Gal4对复制蛋白的影响。目的2将测试起始位置、方向、重复序列组成以及分叉稳定蛋白对(CTG7CAG)n和(ATTCT7AGAAT)n微卫星在复制过程中扩张或收缩的影响。在目标3中，一个来自人类PKD1/TSC2基因座的自然存在的不对称多聚嘧啶序列，我们已经证明形成了一个自然停滞的复制叉子，将从异位的c-myc起源复制，复制体，DNA应激反应蛋白，转位酶和解旋酶在叉子稳定和重新启动中的作用将通过siRNA击倒，芯片和新生DNA的qPCR来评估。我们预计，这些研究将为人类染色体环境中复制启动、复制分叉进展和基因组稳定的过程提供新的见解。与公共卫生相关：对DNA复制启动的不当调控可能会导致染色体断裂、细胞异常分裂和人类疾病。我们已经开发了一种新的系统，可以模拟在癌症和神经退行性疾病中观察到的基因组不稳定。因此，我们的研究很可能为DNA复制的机制、遗传性疾病的起源以及疾病治疗的新模式提供基本的新见解。

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.