Mechanisms Regulating DNA Replication in the Developing Vertebrate Embryo

摘要 在展示各个复制叉的启动方式方面不断取得巨大进步 下一个前沿是了解DNA复制如何与转录协调 和染色质结构。特别是，对DNA的机制和功能知之甚少 复制控制在脊椎动物的发展，当转录和染色质结构， 非常有活力。这种知识差距是一个重要问题，因为在填补这一差距之前， 与表观遗传或DNA相关的发育障碍和癌症中的DNA复制 复制放松管制在很大程度上是不可理解的。它有独特的能力， 传统的细胞培养以及斑马鱼胚胎作为模型系统，我们是独一无二的， 定义调节复制叉启动的关键因素。在这个应用程序中，我们 通过设备补充申请资金，以购买更换生物安全柜， 可用于NIH GM 121703的目的。现有的生物安全柜已有35年以上的历史， 已经过时了目标3中的所有实验都需要使用BSL 2生物安全柜。这 目的是测试乙酰化染色质的早期复制是否依赖于一个 必需复制因子TICRR和表观遗传阅读蛋白BRD 4。这一目标的成果 显示TICRR-BRD相互作用破坏导致复制起始的异常定时 在S阶段。为了解决特定的复制起始蛋白如何确保大的基因组 片段在正确的时间复制，工程化的内源性标记的细胞系的额外的 将使用复制因子。GM 121703研究的成功具有重要意义 因为它试图回答关于DNA复制如何以及为什么开始的基本问题 在整个发展过程中的变化。

Abstract Great strides are continually made in showing how the initiation of individual replication forks is regulated, and the next frontier is to understand how DNA replication is coordinated with transcription and chromatin structure. In particular, little is known about the mechanisms and function of DNA replication control during vertebrate development, when transcription and chromatin structure are highly dynamic. This gap in knowledge is an important problem because, until it is filled, the roles for DNA replication in developmental disorders and cancers associated with epigenetic or DNA replication deregulation will be largely incomprehensible. With the unique capability of using both traditional cell culture as well as zebrafish embryos as model systems, we are uniquely poised to define the key factors in the regulation of replication fork initiation. In this application, we are requesting funds through an equipment supplement to purchase a replacement biosafety cabinet that can be used with the Aims of NIH GM121703. The existing biosafety cabinet is over 35 years old and has become obsolete. All experiments in Aim 3 require the use of a BSL2 biosafety cabinet. This Aim tests whether early replication of acetylated chromatin depends on the physical interaction of an essential replication factor, TICRR, and the epigenetic reader protein BRD4. Results from this aim show that disruption of the TICRR-BRD interaction leads to aberrant timing of replication initiation during S-phase. To address how specific replication initiation proteins ensure that large genomic segments are replicated at the correct time, engineered endogenously tagged cell lines of additional replication factors will be used. Successful achievement of the research in GM121703 is significant because it seeks to answer fundamental questions about how and why DNA replication initiation changes throughout development.