Investigating a novel role for Top2a in R-loop resolution and in YM155 mechanism of action

R环是当新转录的RNA与其互补DNA模板结合时形成的DNA：RNA杂交物， 防止两条DNA链的再退火。R-环是自然发生的，在正常生理过程中发挥作用 免疫球蛋白类转换、转录调控和线粒体复制等过程 入会仪式。病理性R环堆积也在疾病状态下被证实，并与 DNA损伤和基因组不稳定。作为正常细胞的一部分，多种因素参与了R环的分解 生理学包括信使核糖核酸结合蛋白、解旋酶、RNAseH以及拓扑异构酶1和3b。 根据研究小分子YM155治疗间变性甲状腺癌的初步数据， 本项目将研究两个假说-拓扑异构酶2α(Top2α)在解析过程中起到新的作用 其中TOP2α抑制和R环蓄积对YM155的作用机制具有重要意义 它将在两个目标中进行测试：1)评估Top2α在R-环解析中的作用；2)调查Top2在R-环分辨中的作用 研究了TOP2、α和R环堆积在YM155中的作用机制。R环形成(免疫荧光)， 将测量DNA损伤(彗星分析和p-H_2AX焦点)和复制分叉停滞(DNA纤维分析) 在Top2α被击倒之后。TOP2α与YM155的相互作用将通过位点定向的方法进行表征 诱变。RNAseH是DNA：RNA杂交体中RNA所特有的，经常用于确认R-环的积累。 我们将看看RNaseH1过表达是否将细胞从TOP2YM155基因敲除和α治疗中拯救出来。目前， 目前尚不清楚Top2α在R-环分辨中的作用。虽然R环被认为与癌症有关，但几乎没有 知道增加的R环堆积是否可以转化为一种治疗策略。这个项目将 进一步了解R环生物学和癌症病理生理学。 该提案还描述了一项针对医学博士研究生的综合研究和临床培训计划。活动 这项建议包括实验室方法的开发、第一作者的发表和演示技能，包括 机构研讨会和国家科学会议，以及教授研究生保健项目的学生。 学生还将在研究设计、统计和数据解释方面获得宝贵的经验 通过参加文献回顾俱乐部和研究生课程进行评估。临床培训赞助商是 在学生感兴趣的专业中确定有机会进行临床指导和观察， 研究，以及指导指导，以促进过渡到以研究为导向的住院医师计划。 研究和临床培训将在路易斯安那州立大学健康科学中心进行- 什里夫波特(LSUHSC-S)。S有医学院和研究生院，教职员工从事研究工作。 在基础科学和临床学科方面。S为调查人员提供了各种资源，包括专门的 实验室空间和研究核心设施，以协助最新技术以及研讨会、焦点小组、 以及编写赠款、进行研究、生物统计和数据分析方面的专业发展讲习班。

R-loops are DNA:RNA hybrids formed when newly transcribed RNA binds to its complementary DNA template, preventing reannealing of the two DNA strands. R-loops occur naturally and play roles in normal physiological processes including immunoglobulin class switch, transcription regulation, and mitochondrial replication initiation. Pathologic R-loop accumulation has also been demonstrated in disease states and has been linked to DNA damage and genome instability. Various factors are involved in resolving R-loops as part of normal cellular physiology including mRNA binding proteins, helicases, and RNaseH as well as topoisomerases 1 and 3b. Based on preliminary data investigating YM155, a small molecule, in the treatment of anaplastic thyroid cancer, this project will investigate two hypotheses - Topoisomerase 2α (Top2α) plays a novel role in the resolution of R-loops, and Top2α inhibition and R-loop accumulation are important for YM155 mechanism of action which will be tested in two aims: 1) to assess the role of Top2α in R-loop resolution and 2) to investigate the role of Top2α and R-loop accumulation in YM155 mechanism of action. R-loop formation (immunofluorescence), DNA damage (COMET assay and p-H2AX foci), and replication fork stalling (DNA fiber assay) will be measured after Top2α knockdown. The interaction between Top2α and YM155 will be characterized via site-directed mutagenesis. RNaseH is specific to RNA in DNA:RNA hybrids and is often used to confirm R-loop accumulation. We will see if RNaseH1 overexpression rescues cells from Top2α knockdown and YM155 treatment. Currently, there is no known role for Top2α in R-loop resolution. While R-loops have been implicated in cancer, little is known about whether increased R-loop accumulation can be turned into a therapeutic strategy. This project will further understanding of R-loop biology as well as cancer pathophysiology. This proposal also describes an integrated research and clinical training plan for an MD-PhD student. Activities under this proposal include development of lab methods, first author publication, and presentation skills including institutional seminars and national scientific meetings, and teaching students in graduate healthcare programs. The student will also gain valuable experience in research design, statistics, and data interpretation and evaluation by participating in literature review clubs and graduate coursework. A clinical training sponsor is identified in the student’s specialty of interest with opportunities for clinical instruction and observation, for clinical research, and for mentored guidance to facilitate transition into a research-oriented residency program. Research and clinical training will be performed at Louisiana State University Health Sciences Center – Shreveport (LSUHSC-S). LSUHSC-S has a medical school and graduate school with faculty performing research in basic science and clinical disciplines. LSUHSC-S has various resources for investigators including dedicated lab space and research core facilities to assist with the latest technologies as well as seminars, focus groups, and professional development workshops in grant writing, conduct of research, biostatistics and data analysis.