Role of Msh2-Msh3 in determining outcomes in pathways of DNA metabolism

当细胞复制其基因组时，DNA错配修复（MMR）途径起着拼写检查的作用，防止错误，从而减少突变，增加基因组的稳定性。然而，在特定的DNA序列，特别是重复元素的存在下，错配修复活动被改变。人们对这些变化知之甚少，它们促进而不是阻止突变，损害了基因组的稳定性。该项目将确定错配修复蛋白行为中的关键分子变化，这些变化导致基因组从稳定转向不稳定。本科生和研究生将在这个项目中使用尖端的遗传学、分子生物学和生物化学技术进行培训。该项目还将涉及布法罗公立学校的高中生，学生人数约80%是黑人，土著或有色人种。这些活动将促进STEM教育，鼓励人们对科学职业的认识。该项目还将让K-12年级的学生参加课堂研讨会，并让公众参加与科学相关的活动，如基因组日。尽管MMR已被很好地表征，但我们对适当的和异常的msh2 - msh3介导的MMR的机制理解存在关键空白，这两种机制分别导致复制错误纠正和三联体核苷酸重复（TNR）扩增。这项研究将解决关于这两种途径的未解之谜。Msh2-Msh3与MMR中的插入/缺失环和TNR扩增中的DNA二级结构结合，表明不同的DNA结构导致Msh2-Msh3介导的结果不同，并导致基因组的稳定性或不稳定性。目的包括确定Msh2-Msh3与MMR或TNR DNA结构结合时的变构变化，使用交联质谱（XL-MS），以及在MMR或TNR DNA结构存在时确定Msh2-Msh3与下游MLH蛋白复合物之间的相互作用界面。遗传学方法还将用于确定xml - ms鉴定的蛋白质区域的功能相关性。体内和体外方法的结合将提供对影响msh2 - msh3介导的基因组稳定性和不稳定性之间平衡的背景的更全面的看法。这些结果将有助于我们理解MMR通过Msh2-Msh3和MLH复合物的起始和信号传导，以及它如何在存在替代DNA结构的情况下走上错误的路径。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

The DNA mismatch repair (MMR) pathway functions as a spellcheck when cells make copies of their genome, preventing errors and thereby reducing mutations and increasing the stability of the genome. However, in the presence of specific DNA sequences, particularly repetitive elements, mismatch repair activities get altered. These changes, which are poorly understood, promote mutations instead of preventing them and compromise genome stability. This project will identify key molecular changes in the behavior of mismatch repair proteins that lead to the switch from genome stability to instability. Undergraduate and graduate students will be trained in this project using cutting-edge genetics, molecular biology, and biochemical techniques. The project will also involve high school students from Buffalo Public Schools, with a student population that is ~80% black, indigenous or people of color. The activities will promote STEM education and encourage awareness of careers in science. The project will also engage K-12 students in classroom workshops and the general public in science-related events, such as Genome Day.Although MMR is well-characterized, there are key gaps in our mechanistic understanding of appropriate and aberrant Msh2-Msh3-mediated MMR, which result in replication error correction and triplet nucleotide repeat (TNR) expansion, respectively. This research will address unanswered questions about both pathways. Msh2-Msh3 binds to insertion/deletion loops in MMR and to DNA secondary structures in TNR expansions, indicating that distinct DNA structures lead to the differences in Msh2-Msh3-mediated outcomes and result in genome stability or instability. The aims include identifying allosteric changes within Msh2-Msh3 upon binding to MMR versus TNR DNA structures, using cross-linking mass spectrometry (XL-MS), and identifying interaction interfaces between Msh2-Msh3 and downstream MLH protein complexes in the presence of MMR or TNR DNA structures. Genetic approaches will also be used to determine the functional relevance of protein regions identified by XL-MS. The combination of in vivo and in vitro approaches will provide a fuller view of the context that influences the balance between Msh2-Msh3-mediated genome stability and instability. The outcomes will contribute to our understanding of MMR initiation and signaling via Msh2-Msh3 and MLH complexes and how it can go on the wrong path in the presence of alternative DNA structures.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.