MPS-BIO: Replication Conflict and Restart: Resolving Replisome Dynamics in Single Cells

智力优势：细胞分裂需要基因组DNA的复制，由复制机制（复制体）完成。DNA中的断裂、DNA结合蛋白的存在和转录可以阻断复制体，导致冲突，导致基因组不稳定、突变和不能分离完全复制的基因组。虽然很明显，复制暂停作为这些冲突的结果，在这些中断的复制体的命运是未知的。这项研究计划的总体目标是通过合作，跨学科的研究，利用蛋白质组学以及高分辨率单细胞显微镜和数学建模来了解活跃和停滞的复制体。这项研究将定量表征体内复制冲突过程中复制体的动态和蛋白质组成。该项目在枯草芽孢杆菌中解决了这些问题，枯草芽孢杆菌是一种遗传上易于处理的细菌模型系统，它保持了真核复制的一个关键特征：不同的前导链和滞后链聚合酶。该研究将使用新一代荧光显微镜技术，利用细菌细胞的小尺寸，可以观察，计数和跟踪活细胞中的单个蛋白质。具体来说，该项目将确定活跃和停滞复制体的蛋白质组成，无论是在人口水平和单蛋白，单叉，单细胞分辨率。此外，将确定停滞和活跃复制叉处的复制体组分的化学计量和稳定性。总而言之，这项工作将使我们更深入地了解复制体如何在B中面对障碍忠实地复制基因组。更广泛的影响：这项研究项目将影响和推进我们对DNA复制和修复的理解，不仅在革兰氏阳性细菌中，而且在普遍情况下，因为这些过程的许多关键方面在物种间是保守的。此外，这项研究将实施与本科教育紧密结合的跨学科方法。PI将招募有才华的初中，高中和本科生，包括来自代表性不足群体的学生，以及两年制大学的转学生，参加与该项目有关的研究实习。这两个PI都特别致力于促进女性和贫困学生的成功，让她们在教育的早期参与研究。最后，PI致力于为微生物学系建立定量成像基础设施，并继续开发年度成像训练营课程，以培训生物学家在定量成像技术方面的多个级别。

Intellectual Merit: Cell division requires the replication of the genomic DNA, accomplished by the replication machinery (the replisome). Breaks in the DNA, the presence of DNA binding proteins, and transcription can block the replisome, leading to conflicts that result in genomic instability, mutations, and a failure to segregate fully replicated genomes. Although it is clear that replication pauses as the result of these conflicts, the fate of the replisome during these interruptions is unknown. The overall goal of this research plan is to understand both active and stalled replisomes through a collaborative, interdisciplinary study that takes advantage of proteomics as well as high-resolution single-cell microscopy and mathematical modeling. This research will quantitatively characterize the dynamics and protein composition of the replisome during replication conflicts in vivo. The project addresses these questions in Bacillus subtilis, a genetically tractable bacterial model system that maintains a key feature of eukaryotic replication: distinct leading and lagging strand polymerases. The research will use a new generation of fluorescence microscopy techniques that exploit the small size of the bacterial cell and can observe, count, and track single proteins in living cells. Specifically, this project will determine the protein composition of active and stalled replisomes, both at the population level and at single-protein, single-fork, and single-cell resolution. Furthermore, the stoichiometry and stability of the replisome components at stalled and active replication forks will be determined. Altogether, this work will lead to a deeper understanding of how the replisome faithfully duplicates the genome, in the face of obstacles, in B. subtilis.Broader Impacts: This research project will impact and advance our understanding of DNA replication and repair in not only Gram-positive bacteria but universally since many key aspects of these processes are conserved across species. Furthermore, this research will implement an interdisciplinary approach that is tightly integrated with undergraduate education. The PIs will recruit talented middle-school, high-school and undergraduate students, including students from under-represented groups, as well as transfer students from two year colleges, for research internships related to this project. Both PIs are especially dedicated to promoting the success of female and underprivileged students by involving them in research early in their education. Lastly, the PIs are committed to building quantitative imaging infrastructure for the Microbiology Department and to continuing the development of a yearly imaging bootcamp course for training biologists at multiple levels in quantitative imaging techniques.