Chromatin-mediated mechanisms of genome integrity

摘要 我们的研究重点是阐明fl中局部染色质环境对DNA的作用机制。 模板化过程包括DNA复制、转录和DNA修复。虽然取得了相当大的进展，但 虽然我们对在体外引导DNA复制的机制已经有了了解，但我们对此知之甚少 DNA复制的起始点(起始点)是如何在染色体的背景下选择和调节的。这个 我的研究小组开创的基因组方法为这种机制提供了新的见解 fl中的局部染色质状态和结构(核小体和转录因子的占有率)通过什么起关键作用 在包括酿酒酵母和果蝇在内的多种物种中调节DNA复制程序的步骤。我们 最近开发了一种新的方法来‘足迹’真核基因组-同时揭示基因组- 核小体和较小的DNA结合因子(如起始和转录)对DNA的广泛占据 因素)。与使用一个或两个DefiNed DNA模板的生化重建实验不同，我们能够 全面观察细胞周期调节的染色质变化级联，这些变化发生在每个来源周围 酵母基因组中的复制。我们未来的研究将集中在鉴定和鉴定染色质上 解旋酶负载后启动DNA复制所需的中介事件。我们还将调查如何 染色质结构在整个基因组中重新建立，以保持表观遗传的完整性 DNA复制叉子。DNA复制也是双链断裂(DSB)的一个有效来源，如果不是 修复后，可能会导致基因组不稳定。我们处于独特的地位，可以识别和了解动态 位点特异性fic双链断裂诱导后染色质结构的改变及其同源修复 重组或非同源末端连接。最后，与哈特明克实验室(加州杜克大学)合作 我们正在使用在细胞周期中进行的同步酵母种群来开发强大的统计AP- 使我们能够从染色质占有率模拟细胞周期依赖的基因表达变化的方法 数据。

Abstract Our research is focused on elucidating the mechanisms by which the local chromatin environment influences DNA- templated processes including DNA replication, transcription and DNA repair. While considerable progress has been made in our understanding of the mechanisms that direct DNA replication in vitro, we know very little about how start sites of DNA replication (origins) are selected and regulated in the context of the chromosome. The genomic approaches that my research group have pioneered have provided new insights into the mechanisms by which the local chromatin state and structure (nucleosome and transcription factor occupancy) influences key steps in regulating the DNA replication program in multiple species including S. cerevisiae and Drosophila. We have recently developed a novel approach to `footprint' a eukaryotic genome – simultaneously revealing genome- wide occupancy of DNA for both nucleosomes and smaller DNA binding factors (e.g. initiation and transcription factors). Unlike biochemical reconstitution experiments utilizing one or two defined DNA templates, we are able to comprehensively view the cell cycle regulated cascade of chromatin changes that occur surrounding each origin of replication in the yeast genome. Our future research will focus on identifying and characterizing the chromatin mediated events required for initiation of DNA replication following helicase loading. We will also investigate how chromatin structure is re-established throughout the genome to preserve epigenetic integrity following passage of the DNA replication fork. DNA replication is also a potent source of double-stranded breaks (DSB) which, if not repaired, may lead to genomic instability. We are uniquely positioned to identify and understand the dynamics of chromatin structure following the induction of site-specific DSBs and their subsequent repair by homologous recombination or non-homologous end joining. Finally, in collaboration with the Hartemink laboratory (Duke, CS) we are using synchronous populations of yeast proceeding through the cell cycle to develop robust statistical ap- proaches that will enable us to model cell cycle-dependent changes in gene expression from chromatin occupancy data.