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杂交体， 防止两条DNA链的再退火。R环自然发生，并在正常的生理过程中发挥作用。 包括免疫球蛋白类别转换、转录调节和线粒体复制的过程 入会仪式病理性R环积累也已在疾病状态中得到证实，并与 DNA损伤和基因组不稳定性。各种因素参与解析R环作为正常细胞的一部分。 生理学包括mRNA结合蛋白、解旋酶和RNaseH以及拓扑异构酶1和3b。 根据研究YM 155（一种小分子）治疗甲状腺未分化癌的初步数据， 本项目将研究两种假说--拓扑异构酶2α（Top2α）在拆分中发挥新的作用 Top2α抑制和R环积累对YM 155的作用机制很重要 这将在两个目标中进行测试：1）评估Top2α在R环分辨率中的作用，2）研究Top2α在R环分辨率中的作用。 Top2α和R-loop积累在YM 155作用机制中的作用。R环形成（免疫荧光）， 将测量DNA损伤（COMET测定和p-H2 AX焦点）和复制叉停滞（DNA纤维测定） 在Top2α敲除后。Top2α和YM 155之间的相互作用将通过定点突变来表征。 诱变RNaseH对DNA：RNA杂交体中的RNA具有特异性，通常用于确认R环积累。 我们将观察RNaseH 1过表达是否能从Top2α敲低和YM 155处理中拯救细胞。目前， Top2α在R环分解中没有已知的作用。虽然R环与癌症有关， 关于增加的R-环积累是否可以转化为治疗策略，目前还不清楚。该项目将 进一步了解R环生物学以及癌症病理生理学。 该提案还描述了一个综合的研究和临床培训计划的MD-PhD学生。活动 在这个建议下，包括实验室方法的发展，第一作者出版物，和演讲技巧，包括 机构研讨会和国家科学会议，并在研究生保健计划中教授学生。 学生还将获得研究设计，统计和数据解释方面的宝贵经验， 通过参加文学评论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确定与临床教学和观察的机会，临床 研究，并指导指导，以促进过渡到一个以研究为导向的居住计划。 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）。LSUHSC-S有一个医学院和研究生院，教师进行研究 在基础科学和临床学科。LSUHSC-S为调查人员提供各种资源，包括专门的 实验室空间和研究核心设施，以协助最新的技术以及研讨会，焦点小组， 以及关于赠款撰写、研究、生物统计和数据分析的专业发展讲习班。

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.