Genetics of the formation of repair & recombination foci

描述（由申请人提供）：DNA重排发生在许多基因组损伤的反应中，包括自然辐射和用于抗癌的辐射。不幸的是，这些辐射引起的重排往往会刺激新的肿瘤发生。从酵母菌和细菌等简单生物到人类，细胞通过将检查点、修复和重组蛋白定位到不同的病灶来对诱导的DNA损伤做出反应。这些病灶也在DNA合成和分离过程中自发形成，表明细胞在正常细胞周期中处理DNA损伤。这些自发病灶可能代表了修复复制错误和其他对染色体DNA的自然损伤的尝试。细胞也“适应”持续的DNA损伤，即使在没有成功修复的情况下也会尝试分裂。我们以酿酒酵母为模型系统，通过研究修复/重组灶的形成和拆卸，研究了这些过程的遗传控制。我们将继续使用多标记细胞成分的延时显微镜在活酵母细胞中检查这些过程。我们将使用我们设计的试剂来剖析负责焦点形成的遗传途径。我们将利用我们最近开发的高通量方法，使我们能够快速筛选非必需的酵母基因，并识别那些调节DNA修复病灶的组装和拆卸的基因。我们将应用这些相同的方法来发现着丝点组分如何与DNA损伤反应相结合，从而在损伤后以及孢子形成后独特的不对称细胞分裂期间影响病灶。我们的研究将允许进一步了解细胞对电离辐射和自发病变反应的关键成分的体内作用。我们的具体目标如下：(1)利用荧光蛋白标签对染色体双链断裂位点和修复蛋白进行特异性标记，继续对检查点和修复病灶进行分子和遗传学解剖，利用活细胞成像和延迟显微镜技术探索病灶的组成及其组装/拆卸动力学。(2)确定细胞开始修复双链断裂时发生的事件顺序，以解决细胞如何决定形成焦点并确定用于修复的位点。(3)利用酵母基因破坏文库寻找影响DNA修复中心的时间、进程、组装和拆卸的基因，使用高通量筛选能力，包括一种新的交配方法，称为选择性倍体消融以及新的成像平台。(4)检查纺锤体组装检查点的影响，更广泛地说，是着丝点对DNA损伤反应的影响。更新中概述的方法是一般性的。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。由于我们是少数几个致力于研究修复中心（foci）和DNA损伤反应（repair）之间关系的实验室之一，因此我们有意将这一建议集中在DNA损伤反应的遗传学和细胞生物学上。

DESCRIPTION (provided by applicant): DNA rearrangements occur in response to many genomic insults including natural radiation as well as radiation administered to fight cancer. Unfortunately, these radiation-induced rearrangements can often stimulate new neoplasias. Cells ranging from simple organisms such as yeast and bacteria up to humans respond to induced DNA damage by localizing checkpoint, repair and recombination proteins to distinct foci. These foci also form spontaneously during DNA synthesis and segregation suggesting that cells deal with DNA damage during normal cell cycles. These spontaneous foci likely represent attempts to repair replication errors and other natural insults to chromosomal DNA. Cells also "adapt" to persistent DNA damage and attempt to divide even in the absence of successful repair. We have been examining the genetic control of these processes by studying the formation and disassembly of repair/recombination foci using Saccharomyces cerevisiae as a model system. We will continue to examine these processes in living yeast cells using time-lapse microscopy of multi-labeled cellular components. We will use reagents that we have engineered to dissect the genetic pathways responsible for focus formation. We will take advantage of high-throughput methods that we recently developed that allow us to rapidly screen the non-essential yeast genes and identify those that regulate both assembly and disassembly of DNA repair foci. We will apply these same methods to discover how kinetochore components interface with the DNA damage response to affect foci both after damage as well as during a unique asymmetric cell division after spores are formed. Our studies will permit a further glimpse into the in vivo action of key components of the cellular response to both ionizing radiation as well as spontaneous lesions. Our specific aims are as follows: (1) Continue our molecular and genetic dissection of checkpoint and repair foci using fluorescent protein tags to specifically label chromosomal double-strand break sites and repair proteins to explore the composition of foci, their dynamics of assembly/disassembly by using live cell imaging and time-lapse microscopy. (2) Determine the order of events that take place as the cell begins to repair a double-strand break to address how the cell makes the decision to form a focus and identify the locus to be used for repair. (3) Use the yeast gene disruption library to find genes affecting the timing, progression, assembly and disassembly of DNA repair centers using high throughput screening capabilities comprised of a novel mating approach, called selective ploidy ablation along with a new imaging platform. (4) Examine the effects of the spindle assembly checkpoint and, more broadly, the kinetochore on the DNA damage response. The approaches outlined in this renewal are general. The insights that we gain and the methods we develop will not be confined to yeast alone, but will be applicable to many cellular systems. We purposely centered this proposal on the genetics and cell biology of the DNA damage response as we are one of the few labs committed to examining the relationship between repair centers (foci) and the DNA damage response (repair). PUBLIC HEALTH RELEVANCE: DNA damage not only causes cancer, it is used extensively to treat it using radiation therapy and/or chemotherapeutic drugs. Model systems have provided a glimpse into the mechanisms responsible for the repair of DNA damage. In this proposal, we will explore the genetic and cell biological control of DNA repair processes.