Structural studies of DNA at replication fork junctions by 2D fluorescence spectroscopy

生命过程化学（CLP）计划的这一奖项支持了安德鲁·H.马库斯在俄勒冈州大学，以确定在复制叉连接的DNA碱基和主链的局部构象。这些连接是操纵基因的蛋白质的潜在结合位点。目前有一些技术可以提供详细的结构信息，蛋白质-DNA复合物，特别是那些附近的DNA叉路口。该项目正在开发结合联合收割机两种荧光测量的方法，以专门研究分叉连接处的DNA-蛋白质结构动力学。这些方法在理解DNA生物化学的许多过程中具有潜在的普遍用途。此外，这种方法可能有助于识别与人类疾病有关的DNA复制、修复和细胞周期控制复合物功能的缺陷。这些工具随时可供科学界使用。本科生，研究生和博士后学生受益于跨学科的培训和与具有各种科学背景的研究人员的互动。 该研究项目使用二维荧光光谱（2DFS）来研究模型DNA复制叉和功能性复制蛋白-DNA复合物内预选位点的局部DNA碱基和骨架构象以及构象动力学。实验确定的关键结构的最终状态和DNA-蛋白质结合识别基序的复制复合物的形成很重要。该方法采用含有两个相邻定位的荧光碱基类似物探针的DNA构建体。在单独的实验中，在DNA和蛋白质透明的光谱区域吸收的花青发色团被掺入糖-磷酸骨架中。二聚体探针残基的电子状态之间的耦合产生用于推断局部核酸碱基或骨架构象的特征光谱特征。获得的动力学信息是理解蛋白质-DNA复合物组装以执行其专门功能的机制的核心。该项目的第二个目标是开始开发单分子2DFS，以亚秒级的时间分辨率监测局部DNA构象动力学。

This award from the Chemistry of Life Processes (CLP) program supports work by Professor Andrew H. Marcus at the University of Oregon to determine the local conformations of DNA bases and backbones at replication fork junctions. These junctions are potential binding sites for proteins that manipulate genes. There are currently few techniques available that can provide detailed structural information about protein-DNA complexes, particularly those near DNA fork junctions. This project is developing methods that combine two fluorescence measurements, to specifically investigate DNA-protein structural dynamics at fork junctions. The methods are potentially of general use in understanding many processes of DNA biochemistry. In addition, such methods could potentially help to identify defects in the function of DNA replication, repair and cell cycle control complexes involved in human diseases. The tools are made readily available to the scientific community. The undergraduate, graduate and postdoctoral students are benefitting from cross-disciplinary training and interactions with researchers that have a variety of scientific backgrounds. The research project uses two-dimensional fluorescence spectroscopy (2DFS) to investigate the local DNA base and backbone conformations and conformational dynamics at pre-selected sites within model DNA replication forks, and functional replication protein-DNA complexes. The experiments determine key structural end-states and DNA-protein binding recognition motifs important for the formation of replication complexes. The approaches employ DNA constructs containing two adjacently positioned fluorescent base analogue probes. In separate experiments, cyanine chromophores which absorb in a spectral region where DNA and proteins are transparent, are incorporated into the sugar-phosphate backbone. Coupling between the electronic states of the dimer probe residues produces signature spectral features that are used to infer local nucleic acid base or backbone conformation. The acquired information on dynamics is central to understanding mechanisms by which protein-DNA complexes assemble to carry out their specialized functions. A second goal of the project is to begin to develop single-molecule 2DFS to monitor local DNA conformational dynamics with sub-second time resolution.This project is jointly funded by the Chemistry of Life Processes Program in the Division of Chemistry and the Molecular Biophysics Cluster in the Division of Molecular and Cellular Biosciences.