Chemistry and Mechanism of Direct DNA Repair Proteins

描述（由申请人提供）： 由于存在未修复的DNA损伤而导致的遗传变化的积累可能导致癌症和其他疾病的发展。几乎所有的生物都进化出了微妙的系统来定位和修复这些DNA损伤。该研究计划旨在了解两个直接DNA脱烷基化修复蛋白家族的基本机制。化学合成，蛋白质生物化学，大分子X射线晶体学，和各种光谱/物理技术将被用来阐明的DNA碱基修复蛋白O 6-烷基鸟嘌呤-DNA烷基转移酶（AGT）和AlkB的机制。这些蛋白质在保护基因组完整性方面起着至关重要的作用。人AGT蛋白的修复活性也是肿瘤对各种烷基化化疗的抗性的主要因素。AlkB代表了一种新的DNA修复功能，刚刚被发现。该蛋白质家族通过使用新的氧化脱烷基化机制修复烷基化碱基损伤。细菌和人类AlkB蛋白的结构和机制仍然是未知的。AGT和AlkB都与DNA形成不稳定的复合物，这显著阻碍了表征这些蛋白质的蛋白质/DNA相互作用的努力。这里提出的化学策略，以稳定或陷阱的AGT和AlkB的特异性和非特异性蛋白质/DNA复合物的结构表征。设计了DNA探针，合成了探针并用于研究AlkB的作用机制。将采用各种物理和生物化学方法来表征AlkB的铁（II）中心。我们的目标是充分阐明这些蛋白的损伤搜索，识别和修复机制。此外，将开发针对人AGT的更有效的抑制剂，AGT是提高抗癌治疗效率的经证实的靶点。这项研究计划的成功将大大推进对这两个蛋白质家族的理解，并为其他修复系统提供一般机制的影响。

DESCRIPTION (provided by applicant): Accumulation of genetic changes due to the presence of unrepaired DNA lesions can lead to the development of cancer and other diseases. Almost all organisms have evolved delicate systems to locate and repair these DNA lesions. This research program aims to understand the fundamental mechanisms of two direct DNA dealkylation repair protein families. Chemical synthesis, protein biochemistry, macromolecule X-ray crystallography, and various spectroscopic/physical techniques will be employed to elucidate the mechanism of the DNA base repair proteins O6-alkyguanine-DNA alkyltransferases (AGT) and AlkB. These proteins play vital roles in protecting genome integrity. The repair activity of the human AGT protein is also a major factor in tumor resistance to various alkylating chemotherapies. AlkB represents a new type of DNA repair function that has just been discovered. This family of proteins repairs alkylated base lesions by using a novel oxidative dealkylation mechanism. The structure and mechanism of the bacterial and human AlkB proteins remain mostly unknown. Both AGT and AlkB form unstable complexes with DNA, which significantly hampers efforts to characterize the protein/DNA interaction of these proteins. Proposed here are chemical strategies to stabilize or trap both specific and non-specific protein/DNA complexes of AGT and AlkB for structural characterization. DNA probes have been designed which will be synthesized and used to study the mechanism of AlkB. Various physical and biochemical methods will be employed to characterize the iron(ll) centers of AlkB. Our goal is to fully elucidate the damage-searching, -recognition and -repair mechanisms of these proteins. In addition, more potent inhibitors will be developed for human AGT, a proven target for improving the efficiency of anticancer treatments. The success of this research program will significantly advance the understanding of these two protein families and provide general mechanistic implications for other repair systems.