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，但对放疗的耐药性限制了其疗效。红外诱导DNA双链断裂（DSBs），可导致毒性染色体重排；因此，限制这些重排可能对抗辐射至关重要。这种红外诱导的染色体重排可能通过末端连接（EJ）形成，在修复过程中使用不正确的DSB末端。因此，我们的长期目标是确定在EJ过程中限制重排的因素，从而开发肿瘤放射增敏的治疗靶点。为了实现这一目标，我们开发了一种独特的测定方法来量化多个dsb EJ期间正确端与不正确端的使用。使用具有两个串联DSB的染色体报告器，以及我们产生位点特异性非内聚DSB的技术创新，我们可以测量使用单个DSB侧面近端与使用两个DSB远端的EJ。在EJ期间远端使用是不正确的，因为它会导致删除重排。使用该系统，我们发现DNA损伤反应因子RAD50和DNA- pkcs对限制EJ期间不正确的最终使用很重要。由于这些因素也促进了辐射抗性，我们建议验证我们的中心假设，即RAD50和DNA-PKcs在限制EJ期间不正确的最终使用中的作用对细胞辐射抗性至关重要。这一假设的一个推论是，在EJ期间，这些因素在限制不正确的最终使用方面的作用是放射致敏的目标。目标1:

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.