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

项目概要/摘要 候选人寻求通过设计的计划获得额外的研究和职业发展培训 解决她目前专业知识的缺陷。该计划将建立在她现有的技能和知识的基础上 她正在研究一个新系统，并将其带入她职业生涯的下一阶段。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 独立并建立成功的研究计划。 拟议的研究将使用尖端的单分子和计算技术来研究 DNA 解旋复合物 TFIIH 的酶活性。 TFIIH 为转录和 DNA 打开 DNA 修复，但利用两个具有不同机制的独立解旋酶结构域来进行修复。该提案采用了 结合单分子磁力镊子研究和分子动力学模拟： 表征 TFIIH 各个解旋酶结构域的活性（目标 1）；调查两者的影响 完整 TFIIH 复合物中的解旋酶相互连接（目标 2）；并探讨DNA偏好的差异 两种解旋酶以及导致其中一种解旋酶相对于另一种解旋酶具有优势的 DNA 结构方面（目标 3）。 该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 连接这两条途径的方式来修复。拟议研究的结果 将为进一步研究TFIIH活性及其影响因素提供基础，并形成基础 候选人的第一个 R01 提案。 K22 奖项将为候选人提供通过额外的机会发展新研究技能的机会 指导阶段的培训，该培训将由 NHLBI 校内研究部门资助。这些 技能包括构建组合磁力镊子/单分子荧光显微镜， 单分子实验 DNA 底物设计策略和数据分析方法。 在该奖项的指导阶段，她还将参加旨在传授知识的课程和研讨会 管理和教学技能并监督学士学位后实习生。她将参加有关 手稿准备并以她当前的出版记录为基础。培训计划还包括职业生涯 发展活动，例如工作演讲练习、模拟面试和资助写作研讨会。这些 活动将为她提供宝贵的经验和技能，她将利用这些经验和技能作为独立的人 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.