Single molecule studies of the dual-helicase activity of transcription factor TFIIH

项目摘要/摘要 应聘者寻求通过设计的计划获得额外的研究和职业发展培训 以解决她目前专业知识上的不足。该计划将建立在她现有的技能和知识的基础上 研究一种新的系统，她将把它带到她职业生涯的下一个阶段。她将使用 该奖项的指导阶段，旨在培养仪器设计、人员管理和手稿方面的技能 开发和出版。这项培训将使她能够获得过渡所需的额外技能 要独立，并建立一个成功的研究计划。 拟议的研究将使用尖端的单分子和计算技术来研究 DNA解离复合体TFIIH的酶活性。TFIIH为转录和DNA打开DNA 修复，但利用两个独立的解旋酶结构域，具有不同的机制来做到这一点。该提案雇用了一名 单分子磁钳研究和分子动力学模拟相结合，以： 表征TFIIH的单个解旋酶结构域的活性(目标1)；调查两者的影响 在完整的TFIIH复合体中相互作用的解旋酶(目标2)；并探索在DNA偏好上的差异 两个解旋酶和导致其中一个对另一个占优势的DNA结构方面(目标3)。 这项研究项目的完成将为转录和核苷酸切除领域做出贡献 通过阐明TFIIH连接这两条路径的方式来修复。拟议研究的结果 将为进一步研究TFIIH活动及其影响因素提供基础，并形成基础 候选人的第一份R01提案。 K22奖将为候选人提供机会，通过额外的 指导阶段的培训，将由NHLBI校内研究部资助。这些 技术包括建造一种组合的磁镊子/单分子荧光显微镜， 单分子实验的DNA底物设计策略和数据分析方法。 在授奖的指导阶段，她还将参加旨在传授的课程和研讨会 管理和教学技能，并指导毕业后的实习生。她将出席以下研讨会： 准备手稿，并以她目前的出版记录为基础。培训计划还包括职业生涯 发展活动，如实习工作会谈、模拟面试和赠款写作研讨会。这些 活动将为她提供宝贵的经验和技能，她将作为一个独立的 调查员。

Project Summary/Abstract The candidate seeks to acquire additional research and career development training through a plan designed to address deficiencies in her current expertise. The plan will build on her existing skills and knowledge in the study of a new system which she will carry with her into the next phase of her career. She will use the mentored phase of this award to build skills in instrument design, personnel management, and manuscript development and publication. This training will enable her to acquire the additional skills necessary to transition to independence and establish a successful research program. The proposed research will use cutting edge single-molecule and computational techniques to study the enzymatic activity of the DNA-unwinding complex TFIIH. TFIIH opens DNA for both transcription and DNA repair, but utilizes two separate helicase domains with distinct mechanisms to do so. The proposal employs a combination of single-molecule magnetic tweezers studies and molecular dynamics simulations to: characterize the activity of the individual helicase domains of TFIIH (Aim 1); investigate the impact of the two helicases on each other in the full TFIIH complex (Aim 2); and explore the differences in DNA preference of the two helicases and the aspects of DNA structure that lead to dominance of one over the other (Aim 3). Completion of this research project will contribute to the fields of both transcription and nucleotide excision repair by elucidating the ways in which TFIIH links these two pathways. The results of the proposed research will provide a foundation for further studies of TFIIH activity and the factors that influence it, and form the basis of the candidate's first R01 proposal. The K22 award will provide the candidate with the opportunity to develop new research skills through additional training during the mentored phase, which will be funded by the NHLBI Division of Intramural Research. These skills include construction of a combined magnetic tweezers/single molecule fluorescence microscope, strategies for the design of DNA substrates for single-molecule experiments, and data analysis methods. During the mentored phase of the award she will also attend courses and seminars designed to impart management and teaching skills and supervise a post-baccalaureate trainee. She will attend workshops on manuscript preparation and build on her current publication record. The training plan also includes career development activities such as practice job talks, mock interviews, and grant writing workshops. These activities will provide her with invaluable experience and skills that she will draw on as an independent investigator.