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破坏。因此，复杂的机制已经进化出来，以确保DNA合成在复制起点的准确空间和时间起始，以及复制叉进程中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决分叉运动的阻滞，导致DNA的重排、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癌基因的复制起点5'，并且我们已经证明，作为复制起点的核心c-myc复制子的结构、蛋白质结合和功能在其内源性染色体位点和人类基因组的异位位点是相同的。在本应用中，c-myc复制子将被用作模型哺乳动物复制原点，以研究使原点活性的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的重复DNA序列的复制，这些序列阻碍了复制叉的进展。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的限制可能在其他模型系统中无法重现，因此这是一种创新的方法，可以表征染色质环境中影响复制叉稳定性的多个序列和DNA应激反应蛋白。这三个Aims都将使用位点导向的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物整合到人类基因组的一个独特位点。Aim 1将使用缺失分析，通过定量染色质免疫沉淀（ChIP）鉴定c-myc复制子中的蛋白质结合位点，通过新生DNA的PCR定量（qPCR）确定最小c-myc复制起点，并通过ChIP和新生DNA的qPCR评估Gal4招募复制蛋白对起点的影响。Aim 2将测试起源位置、方向、重复序列组成以及叉稳定蛋白对（CTG7CAG）n和（ATTCT7AGAAT）n微卫星在复制过程中的扩张或收缩的影响。在Aim 3中，来自人类PKD1/TSC2基因座的自然产生的不对称多嘌呤- 7多嘧啶序列将从异位c-myc起源复制，形成一个自然的停顿复制叉，复制体、DNA应激反应蛋白、转位酶和解旋酶在叉稳定和重新启动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为人类染色体环境中复制起始、复制叉进展和基因组稳定的过程提供新的见解。公共卫生相关性：DNA复制起始调控不当可导致染色体断裂、细胞分裂异常和人类疾病。我们已经开发了一种新的系统，模仿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，我们的研究可能会为DNA复制机制、遗传疾病的起源和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.