Genetics of the formation of repair & reombination foci

描述（由申请人提供）：DNA重排是对许多基因组损伤的反应，通常与肿瘤以及其他疾病状态（例如免疫缺陷、地中海贫血等）有关。在从酵母菌和细菌等简单生物到人类的细胞中，重组和修复蛋白在DNA损伤时定位于细胞核中的不同病灶。这些病灶也在DNA合成和分离过程中自发形成。为了研究这些过程的遗传控制，我们将以酿酒酵母为模型系统，研究修复/重组灶的形成和拆卸。我们将使用多标记细胞成分的延时显微镜检查活酵母细胞中的这些过程。我们将设计试剂，使我们能够解剖负责焦点形成的遗传途径。由于这可能是一个与细胞周期协调的过程，我们将特别关注细胞周期检查点蛋白对焦点形成的影响。我们已经开发了有助于我们研究的方法，我们建议开发新的试剂，以进一步了解该系统关键成分的体内作用。我们的具体目标是：(1)使用CFP、YFP和DsRed特异性标记染色体DSB位点和修复蛋白，从分子和遗传上解剖检查点和修复病灶。(2)解决DNA复制、修复和检查点激活的协调问题，这源于我们对dsb修复特异性发生在细胞周期的S期（或G2）的观察。化学制剂以及基因组不稳定突变将被检查。(3)通过延时显微镜、光漂后荧光恢复（FRAP）和光漂中荧光损失（FLIP）分析探讨焦点组装/拆卸的动力学。(4)通过将DsRed与特异性识别DNA损伤诱导的中央修复和重组蛋白表位的抗体融合，开发一种新的方法来可视化体内重要生物事件的时间。这些方法是通用的，我们获得的见解和我们开发的方法将不仅仅局限于酵母，而是适用于许多细胞系统。

DESCRIPTION (provided by applicant): DNA rearrangements occur in response to many genomic insults and are often associated with neoplasia as well as with other disease states (e.g., immuno-deficiencies, thalassemias etc.). In cells ranging from simple organisms such as yeast and bacteria up to humans, recombination and repair proteins localize to distinct foci in the nucleus in response to DNA damage. These foci also form spontaneously during DNA synthesis and segregation. To examine the genetic control of these processes, we will look at the formation and disassembly of repair/recombination foci using Saccharomyces cerevisiae as a model system. We will examine these processes in living yeast cells using time-lapse microscopy of multi-labeled cellular components. We will engineer reagents that will allow us to dissect the genetic pathways responsible for focus formation. As this is likely a coordinated process with the cell cycle, we will pay particular attention to cell cycle checkpoint proteins for their effects on focus formation. We have already developed methods that will aid in our studies and we propose to develop new reagents that will permit a further glimpse into the in vivo action of key components of this system. Our specific aims are: (1) to molecularly and genetically dissect checkpoint and repair foci using CFP, YFP and DsRed to specifically label chromosomal DSB sites and repair proteins.. (2) to address the coordination of DNA replication, repair and checkpoint activation which stems from our observation that repair of DSBs specifically occurs in S phase (or G2) of the cell cycle. Chemical agents as well as genome instability mutants will be examined. (3) to explore the dynamics of focus assembly/disassembly by using time-lapse microscopy, fluorescent recovery after photo bleaching (FRAP) and fluorescent loss in photo bleaching (FLIP) analyses. (4) to develop a new approach to visualize the timing of important biological events in vivo by fusing DsRed to antibodies that specifically recognizes DNA damage-induced epitopes of central repair and recombination proteins. These approaches are general and the insights that we gain and the methods that we develop will not be confined to yeast alone, but will be applicable to many cellular systems.