Correct end use during end joining and radioresistance

描述(由申请人提供)：电离辐射(IR)可导致显著的肿瘤细胞死亡，但对放射治疗的抵抗会限制其疗效。IR诱导DNA双链断裂(DSB)，可导致有毒的染色体重排；因此，限制这些重排可能是辐射抗性的关键。这种IR诱导的染色体重排可能是通过末端连接(EJ)形成的，在修复过程中使用了错误的DSB末端。因此，我们的长期目标是确定限制EJ期间重排的因素，从而开发肿瘤放射增敏的治疗靶点。为了推进这一目标，我们开发了一种独特的方法来量化在多个DSB的EJ过程中正确和不正确的末端的使用。使用带有两个串联DSB的染色体报告程序，以及我们产生特定部位的非结合DSB的技术创新，我们可以测量使用单个DSB两侧的近端的EJ与使用两个DSB的远端的EJ。在EJ期间使用远端是不正确的，因为它会导致删除重排。利用这个系统，我们发现DNA损伤反应因子Rad50和DNA-PKcs对于限制EJ过程中的错误最终使用是重要的。由于这些因素也促进辐射抗性，我们建议检验我们的中心假设，即Rad50和DNA-PKcs在限制EJ期间不正确的终端使用方面的作用对细胞辐射抗性至关重要。这一假设的一个推论是，这些因素在限制EJ期间不正确终端使用方面的功能是放射增敏的靶点。目标1： 为了确定RAD50功能在限制EJ期间不正确的终端使用方面与其在DNA修复中的其他作用相比对辐射抗性的重要性。为此，我们将检测一系列Rad50突变体在Rad50缺陷的人类细胞中补充一组DNA修复功能并促进辐射抗性的能力。目的2：比较DNA-PKcs在DNA修复中的其他作用，确定DNA-PKcs功能在限制EJ过程中不正确的终端使用方面对辐射抗性的重要性。使用与目标1类似的方法，我们将检测一系列DNA-PKcs突变体在DNA-PKcs缺陷的哺乳动物细胞中补充DNA修复功能和促进辐射抗性的能力。目的3.确定增加两个串联DSB之间的距离如何影响EJ过程中不正确的终端使用，以及Rad50和DNA-PKcs限制EJ介导的这种删除重排的相对要求。这些拟议的研究具有重要意义，因为它们将为Rad50和DNA-PKcs如何促进辐射抵抗提供新的见解，这对它们成为肿瘤放射增敏的治疗靶点至关重要。我们的研究具有创新性，因为它将建立一个新的范式来理解Rad50和DNA-PKcs在促进辐射抗性中的作用，也因为我们在目标3中建议开发一个新的报告系统来研究DSB之间的距离如何影响EJ期间的最终使用。我们提出的研究也是创新的，因为它将深入了解EJ期间不正确的终端使用对辐射毒性的重要性，这将导致肿瘤放射增敏的新治疗策略。 公共卫生相关性：拟议的研究与公共健康相关，因为确定DNA修复蛋白如何限制辐射毒性将导致肿瘤放射增敏的新治疗策略。因此，这项拟议的研究支持了美国国立卫生研究院改善癌症治疗结果的使命。

DESCRIPTION (provided by applicant): Ionizing radiation (IR) can cause to significant tumor cell death, however resistance to radiotherapy can limit its efficacy. IR induces DNA double-strand breaks (DSBs) that can lead to toxic chromosomal rearrangements; such that limiting these rearrangements may be critical for radioresistance. Such IR-induced chromosomal rearrangements likely form via end joining (EJ) that uses incorrect DSB ends during repair. Thus, our long-term goal is to define the factors that limit rearrangements during EJ, and thereby develop therapeutic targets for tumor radiosensitization. To advance this goal, we developed a unique assay to quantify the use of correct versus incorrect ends during EJ of multiple DSBs. Using a chromosomal reporter with two tandem DSBs, and our technical innovation of generating site-specific non-cohesive DSBs, we can measure EJ that uses proximal ends that flank a single DSB versus EJ that uses distal ends of two DSBs. Distal end use during EJ is incorrect, since it causes a deletion rearrangement. Using this system, we found that the DNA damage response factors RAD50 and DNA-PKcs are important to limit incorrect end use during EJ. Since these factors also promote radioresistance, we propose to test our central hypothesis that the role of RAD50 and DNA-PKcs in limiting incorrect end use during EJ is critical for cellular radioresistance. A corollary of this hypothesis is that the funcion of these factors in limiting incorrect end use during EJ is a target for radiosensitization. Aim 1: To determine the importance to radioresistance of RAD50 function in limiting incorrect end use during EJ, as compared to its other roles in DNA repair. For this, we will examine a series of RAD50 mutants for the ability to complement a set of DNA repair functions, and promote radioresistance, in RAD50-deficient human cells. Aim 2: To determine the importance to radioresistance of DNA-PKcs function in limiting incorrect end use during EJ, as compared to its other roles in DNA repair. Using a similar approach as Aim 1, we will examine a series of DNA-PKcs mutants for the ability to complement DNA repair functions, and promote radioresistance, in DNA-PKcs-deficient mammalian cells. Aim 3. To determine how increasing the distance between two tandem DSBs affects incorrect end use during EJ, and the relative requirement of RAD50 and DNA-PKcs for limiting such EJ-mediated deletion rearrangements. These proposed studies are significant, because they will provide novel insight into how RAD50 and DNA-PKcs promote radioresistance, which is critical for their development as therapeutic targets for tumor radiosensitization. Our study is innovative because it will establish a new paradigm for understanding the role of RAD50 and DNA-PKcs in promoting radioresistance, and because we propose in Aim 3 to develop a new reporter system to examine how the distance between DSBs affects end use during EJ. Our proposed study is also innovative because it will provide insight into the importance of incorrect end use during EJ for radiation toxicity, which will lead to new therapeutic strategies for tumor radiosensitization. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health, because determining how DNA repair proteins limit radiation toxicity will lead to new therapeutic strategies for tumor radiosensitization. Thus, the proposed study supports the NIH mission for improving patient outcomes from cancer treatment.