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会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。本研究旨在了解两个直接DNA脱烷基修复蛋白家族的基本机制。将利用化学合成、蛋白质生物化学、高分子X射线结晶学和各种光谱/物理技术来阐明DNA碱基修复蛋白O6-烷基鸟嘌呤-DNA烷基转移酶(AGT)和AlkB的作用机制。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。人类AGT蛋白的修复活性也是肿瘤对各种烷基化化疗耐药的主要因素。AlkB代表了一种刚刚被发现的新型DNA修复功能。这一家族的蛋白质通过使用一种新的氧化脱烷基机制来修复烷化碱基损伤。细菌和人类AlkB蛋白的结构和机制大多尚不清楚。AGT和AlkB都与DNA形成不稳定的络合物，这严重阻碍了对这些蛋白质/DNA相互作用的研究。本文提出了稳定或捕获AGT和AlkB的特定和非特定蛋白质/DNA复合体的化学策略，以进行结构表征。设计了DNA探针，并将其用于研究AlkB的作用机理。将使用各种物理和生物化学方法来表征碱性B的铁(11)中心。我们的目标是全面阐明这些蛋白质的损伤搜索、识别和修复机制。此外，还将为人类AGT开发更有效的抑制剂，这是提高抗癌治疗效率的已被证实的目标。这项研究的成功将极大地促进对这两个蛋白质家族的了解，并为其他修复系统提供普遍的机制启示。

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.