G-A MISMATCH RECOGNITION AND REPAIR BY E COLI MUTY

失配修复最近吸引了大量的关注， 发现错配修复酶的突变与 遗传性结肠癌e.大肠杆菌有一个精心设计的防线， 不匹配的有害影响。几十年的工作在E。杆菌 系统已经大大提高了对这些重要的理解 内切酶然而，即使对细菌酶进行了多年的研究， 许多关于DNA修复酶的重要而未解的问题 保持。事实上，影响DNA损伤识别的因素 包括破坏周围的层序环境的影响， 目前仍不清楚.急诊大肠杆菌酶MutY在预防 由于氧化性蛋白质的错误编码而引起的错配引起的突变 DNA损伤。MutY作为腺嘌呤糖基化酶， 腺嘌呤与7，8-二氢-8-氧代鸟嘌呤（OG）配对。 很少 机理研究集中在作用于嘌呤碱基的糖基化酶上。一个 MutY的不寻常之处在于它是一种含有[4Fe-4S]簇的蛋白质 并且与DNA修复酶内切核酸酶III具有显著的同源性。 [4Fe-4S]簇结构域可能代表一种新的 错配识别和修复的基序确实非常有趣。 因此，MutY不仅在保持高保真度方面功能重要， 除了DNA复制外，它还具有独特的结构和机械特性。 拟议研究的目标是提供一个详细的了解 MutY的结构和功能特性。最初的重点 将决定G：A和OG：A失配的性质， 这些底物对于MutY的识别和修复至关重要。在 特别是G：A周围的序列环境的影响 将确定不匹配以及原点的确定 序列依赖效应。含有修饰的G：A和OG：A的底物 将准备确定G/OG上的重要功能组 或有效失配识别所需的A。小说 将制备MutY抑制剂，以提供机制信息 作为酶的生化和光谱研究的工具， 抑制剂复合物 对DNA性质的深入了解 MutY的底物将为随后的研究提供基础， MutY-DNA复合物的性质，特别强调 阐明了酶中不寻常的金属中心的作用。这些 实验将集中在确定[4Fe-4S]簇是否 与错配识别和DNA结合密切相关。

Mismatch repair has recently attracted a great deal of attention due to the discovery that mutations in mismatch repair enzymes are linked to hereditary colon cancer. E. Coli has an elaborate line of defense against the deleterious effects of mismatches. Decades of work on the E. Coli systems has significantly advanced the understanding of these important enzymes. However, even with years of research on the bacterial enzymes, many important and unanswered questions concerning DNA repair enzymes remain. Indeed, the factors influencing the recognition of DNA damage including the effects of sequence environment surrounding the damage are still unclear. The E. Coli enzyme, MutY is important in the prevention of mutations caused by mismatches that arise due to miscoding from oxidative damage to DNA. MutY acts as an adenine glycosylase to remove an undamaged adenine paired with 7,8-dihydro-8-oxoguanine (OG). Very little mechanistic work has focused on glycosylases that act on purine bases. An unusual aspect of MutY is that it is a [4Fe-4S] cluster-containing protein and has significant homology to the DNA repair enzyme, endonuclease III. The possibility that the [4Fe-4S] cluster domain could represent a new motif for mismatch recognition and repair is indeed quite intriguing. Thus, MutY is not only functionally important in maintaining high fidelity DNA replication, it also has unique structural and mechanistic properties. The goal of the proposed research is to provide a detailed understanding of the structural and functional properties of MutY. The initial focus will be determining the properties of G:A and OG:A mismatch containing substrates which are critical for recognition and repair by MutY. In particular, the influence of the sequence environment around the G:A mismatch will be determined as well as the a determination of the origin of sequence dependent effects. Modified G:A and OG:A containing substrates will be prepared to determine the important functional groups on the G/OG or the A that are required for efficient mismatch recognition. Novel inhibitors for MutY will be prepared to provide mechanistic information and as tools for biochemical and spectroscopic studies of the enzyme- inhibitors complex. A solid understanding of the properties of the DNA substrates for MutY will provide the subsequent basis for investigation of the properties of MutY-DNA complexes with a particular emphasis on elucidation of the role of the unusual metal center in the enzyme. These experiments will focus on determining whether or not the [4Fe-4S] cluster is intimately involved in mismatch recognition and DNA binding.