The role of Sen1 in transcription coupled DNA repair (TCR)

DNA修复在维持基因组稳定性中起着至关重要的作用。本项目研究Sen 1蛋白在模式生物芽殖酵母中参与转录偶联DNA修复（TCR）的机制。Sen 1是人senataxin的同源物，senataxin是一种涉及共济失调-眼用不能-2（AOA 2）和青少年肌萎缩性侧索硬化症（ALS 4）的常染色体显性形式的蛋白质。鉴于DNA修复机制是高度保守的，从酵母蛋白的研究中产生的发现可能适用于包括人类在内的其他物种。除了在重要的DNA修复机制方面取得科学进展外，该项目还将对教育产生重大影响，使研究生和本科生能够获得酵母遗传学和分子生物学方面的研究经验和熟练程度。该项目的相关新方法和发现将与科学界分享，并在教室、当地科学博览会以及国家和国际科学会议上展示。DNA修复的TCR途径已被发现近30年。然而，由于其复杂性，特别是在真核细胞中，TCR的潜在机制仍然知之甚少。最近的研究结果表明，Sen 1在这一途径中发挥更直接的作用比众所周知的TCR因子Rad 26。该项目有3个目标，这些目标建立在这一重大发现的基础上。目的1是确定Sen 1与单链DNA内切酶Rad 2和RNA聚合酶II（RNAP II）最大亚基Rpb 1的C末端结构域（CTD）在TCR过程中的相互作用的意义。一种方法，允许明确映射直接蛋白质-蛋白质相互作用的活酵母细胞将被用来确认和系统地定义的相互作用。目的二是确定TCR中Sen 1最小必需区（Sen 1-MER）的功能。除了ATP酶解旋酶活性和核定位功能外，相对较大（约900个氨基酸残基）的Sen 1-MER可与DNA、新生RNA和某些蛋白质相互作用。很可能Sen 1-MER在TCR中也起重要作用。将分离和表征TCR缺陷型Sen 1-MER突变体，并分析Sen 1-MER过表达对TCR的影响。目的3是确定Sen 1和Rad 26如何与TCR阻遏物相互作用以调节DNA损伤的转录旁路并调节RNAP II复合物在DNA损伤处的停滞和/或破坏以进行修复。这项全面的分析有望为Sen 1及其在TCR中的功能提供详细的见解。

DNA repair plays a vital role maintaining genome stability. This project addresses the mechanism of how Sen1 protein is implicated in transcription coupled DNA repair (TCR) in the model organism budding yeast. Sen1 is a homolog of human senataxin, a protein implicated in ataxia-ocular apraxia-2 (AOA2) and an autosomal dominant form of juvenile amyotrophic lateral sclerosis (ALS4). Given the fact that the DNA repair mechanisms are highly conserved, the findings generated from study of the yeast protein are likely to be applicable to other species including human. In addition to scientific advancement on an important DNA repair mechanism, the project will have a significant impact on education, enabling graduate and undergraduate students to acquire research experience and proficiency in yeast genetics and molecular biology. Relevant new methods and findings from the project will be shared with the scientific community and presented in classrooms, local science fairs and at national and international scientific meetings.The TCR pathway of DNA repair has been known for almost 30 years. However, the underlying mechanism of TCR is still poorly understood because of its complexity, especially in eukaryotic cells. Recent findings suggest that Sen1 plays a more direct role in this pathway than the well-known TCR factor Rad26. This project has 3 objectives that build on this significant finding. Objective 1 is to determine the implications of the interactions of Sen1 with Rad2, a single-stranded DNA endonuclease, and with the C-terminal domain (CTD) of Rpb1, the largest subunit of RNA polymerase II (RNAP II), during TCR. A method that allows unambiguous mapping of direct protein-protein interactions in living yeast cells will be utilized to confirm and systematically define the interactions. Objective 2 is to determine the functionality of the minimal essential region of Sen1 (Sen1-MER) in TCR. In addition to its ATPase-helicase activity and nuclear localization function, the relatively large (~ 900 amino acid residues) Sen1-MER may interact with DNA, nascent RNA and certain proteins. It is likely that Sen1-MER also plays an important role in TCR. TCR-deficient Sen1-MER mutants will be isolated and characterized, and the effects of overexpression of Sen1-MER on TCR will be analyzed. Objective 3 is to determine how Sen1 and Rad26 interplay with TCR repressors to regulate transcription bypass of DNA lesions and modulate stalling and/or disruption of the RNAP II complex at DNA lesions for repair. This comprehensive analysis is expected to provide detailed insights into Sen1 and its function in TCR.