G-A MISMATCH RECOGNITION AND REPAIR BY E COLI MUTY

失配修复最近引起了极大的关注，原因是 错配修复酶突变与 遗传性结肠癌。E.Coli有一条精心设计的防线，可以预防 不匹配的有害影响。几十年来对E.Coli的研究 系统极大地促进了对这些重要信息的理解 酵素。然而，即使对细菌酶进行了多年的研究， 关于DNA修复酶的许多重要和悬而未决的问题 留下来。的确，影响DNA损伤识别的因素 包括破坏周围的序列环境的影响是 仍不清楚。大肠杆菌酶MutY在预防肺炎中起着重要作用。 因氧化过程中的编码错误而导致的不匹配导致的突变 对DNA的破坏。MutY作为一种腺嘌呤糖基酶来清除未受损的 腺嘌呤与7，8-二氢-8-氧鸟嘌呤(OG)配对。很少 机械论的工作主要集中在作用于嘌呤碱基的糖基酶上。一个 MutY的不同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，内切酶III有显著的同源性。 [4Fe-4S]簇结构域可能代表一种新的 错配识别和修复的主题确实相当耐人寻味。 因此，MutY不仅在功能上对保持高保真度很重要 除了DNA复制，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的结构和功能性质。最初的焦点 将确定G：A和OG的属性：包含 对于MutY的识别和修复至关重要的底物。在……里面 特别是，G：A周围序列环境的影响 将确定不匹配以及原点的确定 序列依赖效应。改性G：A和OG：A含底物 将准备确定G/OG上的重要官能团 或者是有效的失配识别所需的A。小说 MutY的抑制剂将准备好提供机械信息 作为酶的生化和光谱研究的工具- 抑制剂复合体。对DNA的性质有扎实的了解 MutY的底物将为后续的研究提供基础 MutY-DNA络合物的性质 阐明不寻常的金属中心在酶中的作用。这些 实验将集中在确定[4Fe-4S]团簇是否 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.