Mechanistic Dissection of the BRCA1-SETX-dependent Pathway of R-loop Avoidance and Genome Maintenance

项目摘要 基因组完整性的维持取决于细胞修复受损DNA和解决DNA缺陷的能力。 转录-复制冲突。在这方面，R环，三链核酸结构， 携带与DNA模板杂交的RNA转录物，可以在多个细胞中损害基因组稳定性， 的方式具体地，R环结构内的ssDNA易受核裂解的影响，从而导致 转录相关诱变或转录相关重组。此外，碰撞 具有R环的DNA复制机器的一部分可以导致复制叉崩溃，DNA双链断裂， 断裂（DSB），叉融合和染色体易位，然后可导致肿瘤 转化和肿瘤发生。 我们的MPI补助金的这种竞争性延续利用了我们在DNA修复酶学方面的独特专业知识 和细胞生物学建模来描绘R环解析机制的结构-功能， SF 1家族解旋酶Senataxin（SETX）和肿瘤抑制复合物BRCA 1-BARD 1。在 具体目标1，我们将定义SETX的异常通用的核酸解旋活性，并测试 SETX直接通过特定的解旋活性解决R环的假设。具体目标2 将确定BRCA 1-BARD 1在SETX介导的R环分解中的作用，以检验以下假设： BRCA 1-BARD 1与SETX合作，通过询问SETX来解析病理性R环， BRCA 1-BARD 1在我们重建的生化系统和细胞中。 MPI的更新是基于帕特里克宋博士， 领先的DNA修复酶学家，以及加里库普弗博士，一位利用遗传学 范可尼贫血模型，以促进对DNA修复途径和机制的理解。 与众多重要的合著论文一起，我们持续的合作努力 承诺发挥最大程度的影响，并提供对机械基础的洞察力 与肿瘤抑制途径相关的主要基因组维持途径。

Project Summary Maintenance of genomic integrity depends on the ability of cells to repair damaged DNA and resolve transcription-replication conflicts. In this regard, R-loops, three-stranded nucleic acid structures that harbor an RNA transcript hybridized to a DNA template, can compromise genome stability in multiple ways. Specifically, the ssDNA within the R-loop structure is vulnerable to nucleolytic cleavage, resulting in transcription-associated mutagenesis or transcription-associated recombination. Moreover, collisions of the DNA replication machinery with R-loops can cause replication fork collapse, DNA double-strand breaks (DSBs), fork fusions, and chromosome translocations, which can then lead to neoplastic transformation and tumorigenesis. This competitive continuation of our MPI grant leverages our unique expertise in DNA repair enzymology and cell biology modeling to delineate the structure-function of an R-loop resolution machinery comprised of the SF1 family helicase Senataxin (SETX) and the tumor suppressor complex BRCA1-BARD1. In Specific Aim 1, we will define the unusually versatile nucleic acid unwinding activity of SETX and test the hypothesis that SETX resolves R loops directly through specific unwinding activity. Specific Aim 2 will determine the role of BRCA1-BARD1 in SETX-mediated R-loop resolution to test the hypothesis that BRCA1-BARD1 cooperates with SETX to resolve pathological R-loops by interrogating SETX and BRCA1-BARD1 in our reconstituted biochemical systems and in cells. This MPI renewal is based on the longstanding and productive collaboration between Dr Patrick Sung, a leading DNA repair enzymologist, and Dr Gary Kupfer, a physician-scientist who has utilized the genetic model of Fanconi anemia to advance understanding of DNA repair pathways and mechanisms. Together, with numerous coauthored papers of high significance, our continuing collaborative endeavors promise to exert impact of the highest degree and to provide insight into the mechanistic underpinnings of a major genome maintenance pathway that is linked to tumor suppression pathways.