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

描述（由申请人提供）：本研究的长期目标是阐明DNA修复糖基化酶用于检测和切除基因组DNA中受损碱基的螺旋外碱基识别机制。这项工作涉及DNA修复过程，涉及保持基因组的完整性，这在许多方面影响人类健康，包括预防癌症和遗传性遗传疾病。我们将使用新型核磁共振和晶体学方法来揭示这种称为“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，然后询问，正常的和受损的碱基，从DNA双链体出现，因为热诱导的碱基对呼吸运动，或者，酶是否使用主动机制，以促进受损的碱基从DNA堆栈驱逐。差异是至关重要的。对于前者，受损位点的结构和动力学启动其自身的修复，而对于后者，酶提供了排出碱基的必要手段。具体目标是（i）开发一种称为“反应坐标调谐”的新化学方法，以捕获在尿嘧啶DNA糖基化酶的胸腺嘧啶和尿嘧啶翻转途径上非常早期发生的另外能量不稳定的螺旋外中间体。该结构将揭示酶用于促进碱基翻转的最早的相互作用。（ii）在结构的指导下，定点诱变将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NMR亚氨基质子交换方法进行测量。这些DNA动态测量将阐明碱基对开放的详细机制。(Hi)使用NMR弛豫方法来测量参与螺旋外识别的最早步骤的酶骨架NH基团的动力学。酶的运动必须足够快，以有效地捕获尿嘧啶在其螺旋外状态的寿命。因此，DNA动力学将与酶动力学联系起来。 公共卫生相关性：在人体的每个细胞中，DNA碱基每天都会被破坏数百次。如果没有特殊的酶机器来定位和修复这些受损的部位，癌症和疾病就会猖獗。这项工作的目标是阐明这些机器如何定位这些位点，长期目标是使用识别原理设计分子，有效地将这些酶引导到基因组中特定的受损位点。

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.