Linking Enzyme and DNA Dynamics: The Mechanism of Extrahelical Damaged based dete

描述(申请人提供)：这项研究的长期目标是阐明DNA修复糖基酶用来检测和切除基因组DNA中受损碱基的螺旋外碱基识别机制。这项工作涉及保护基因组完整性所涉及的DNA修复过程，这在许多方面影响人类健康，包括预防癌症和遗传疾病。我们将使用新的核磁共振和结晶学方法来揭示这种被称为“碱基翻转”的识别机制的重要结构和动力学方面。一个关键的目标是确定酶是否被动地捕获并询问由于热诱导的碱基对呼吸运动而从DNA双链中出现的正常和受损的碱基，或者酶是否使用主动机制来促进受损的碱基从DNA堆叠中排出。区别是至关重要的。对于前者，损伤部位的结构和动力学启动了自身的修复，而对于后者，酶提供了弹出碱基的必要手段。具体目标是(I)开发一种名为“反应坐标调节”的新化学方法，以捕获一种能量不稳定的螺旋外中间体，该中间体在尿嘧啶DNA糖基酶翻转胸腺嘧啶和尿嘧啶的途径中很早就出现。该结构将揭示酶用来促进碱基翻转的最早的相互作用，(Ii)在该结构的指导下，定点突变将被用于删除参与稳定螺旋外构象的酶侧链。突变对翻转碱基动力学的影响将使用我们最近开发的核磁共振亚米诺质子交换方法来测量。这些DNA动态测量将阐明碱基对开放的详细机制。(Hi)使用核磁共振弛豫方法测量参与螺旋外识别最早步骤的酶主链NH基团的动力学。酶的运动必须足够快，才能有效地捕获处于非螺旋状态的尿嘧啶。因此，DNA动力学将与酶动力学联系在一起。 与公共卫生相关：人体每个细胞每天都会有数百次DNA碱基遭到破坏。如果没有特殊的酶机器来定位和修复这些受损的部位，癌症和疾病将会猖獗。这项工作的目标是阐明这些机器是如何定位这些位点的，长期目标是利用识别原理设计分子，有效地将这些酶定向到基因组中特定的受损部位。

DESCRIPTION (provided by applicant): The long term objective of this research is to elucidate the extrahelical base recognition mechanism used by DNA repair glycosylases to detect and excise damaged bases in genomic DMA. This work relates to the DNA repair processes involved in preserving the integrity of the genome, which impacts human health in many ways, including the prevention of cancer and inheritable genetic diseases. We will use novel NMR and crystallographic methods to uncover the important structural and dynamic aspects of this recognition mechanism termed "base flipping". A key goal is to determine whether enzymes passively capture, and then interrogate, normal and damaged bases that emerge from the DNA duplex because of thermally induced base pair breathing motions, or alternatively, whether enzymes use active mechanisms to promote damaged base expulsion from the DNA stack. The difference is critical. For the former, the structure and dynamics of the damaged site initiates its own repair, and for the latter, the enzyme provides the essential means for ejecting the base. The specific aims are (i) to develop a new chemical approach called "reaction coordinate tuning" to trap an otherwise energetically unstable extrahelical intermediate that occurs very early on the pathway for thymine and uracil flipping by uracil DNA glycosylase. This structure will uncover the earliest interactions that the enzyme uses to promote base flipping, (ii) Directed by the structure, site- directed mutagenesis will be used to delete enzyme side chains involved in stabilizing the extrahelical conformation. The effects of the mutations on the dynamics of the flipped base will be measured using our recently developed NMR imino proton exchange methods. These DNA dynamic measurements will elucidate the detailed mechanism of base pair opening. (Hi) Use NMR relaxation methods to measure the dynamics of enzyme backbone NH groups involved in the earliest step in extrahelical recognition. The enzyme motions must be rapid enough to efficiently trap the uracil in the life-time of its extrahelical state. Hence, DNA dynamics will be linked to enzyme dynamics. Public Health Relevance: DNA bases are damaged hundreds of time each day in every cell of the human body. Without extraordinary enzyme machines to locate and repair these damaged sites, cancer and disease would be rampant. The goal of this work is to elucidate how these machines locate these sites, with the long term goal of using the recognition principles to design molecules that efficiently direct these enzymes to specific damaged sites in the genome.