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，然而对放射治疗的抵抗可限制其疗效。IR诱导DNA双链断裂（DSB），其可导致毒性染色体重排;因此限制这些重排对于辐射抗性可能是至关重要的。这种IR诱导的染色体重排可能通过在修复期间使用不正确的DSB末端的末端连接（EJ）形成。因此，我们的长期目标是确定在EJ期间限制重排的因素，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测定方法来量化在多个DSB的EJ期间正确末端与不正确末端的使用。使用具有两个串联DSB的染色体报告基因，以及我们产生位点特异性非粘性DSB的技术创新，我们可以测量使用侧接单个DSB的近端的EJ与使用两个DSB的远端的EJ。EJ过程中远端使用不正确，因为它会导致缺失重排。使用该系统，我们发现DNA损伤反应因子RAD 50和DNA-PKcs对于限制EJ期间的不正确最终使用是重要的。由于这些因素也促进了辐射抗性，我们建议测试我们的中心假设，即在EJ过程中，RAD50和DNA-PKcs在限制不正确的最终使用中的作用对细胞的辐射抗性至关重要。这一假设的一个推论是，这些因素在限制EJ期间不正确的最终使用中的功能是放射增敏的目标。目标1： 确定与其在DNA修复中的其他作用相比，RAD50功能在限制EJ期间不正确的最终使用中对辐射抗性的重要性。为此，我们将研究一系列的RAD50突变体的能力，以补充一套DNA修复功能，并促进辐射抗性，在RAD50缺陷的人类细胞。目标二：确定DNA-PKcs功能在EJ期间限制不正确的最终使用中对辐射抗性的重要性，与其在DNA修复中的其他作用相比。使用与目标1类似的方法，我们将研究一系列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。目标3。确定增加两个串联DSB之间的距离如何影响EJ期间的不正确末端使用，以及限制此类EJ介导的缺失重排对RAD50和DNA-PKcs的相对要求。这些拟议的研究是重要的，因为它们将提供新的见解如何RAD50和DNA-PKcs促进辐射抗性，这是至关重要的，他们的发展作为肿瘤放射增敏的治疗靶点。我们的研究是创新的，因为它将建立一个新的范式，了解RAD50和DNA-PKcs在促进辐射抗性的作用，因为我们在目标3中提出开发一个新的报告系统，以检查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性的，因为它将深入了解EJ期间不正确的最终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。

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.