MECHANISTIC STUDIES OF THE FE/S ENZYME SP LYASE

这项建议的具体目的是对孢子光产物(SP)裂解酶(一种依赖于腺苷甲硫酮的铁硫酶)修复DNA的机制有一个了解。将SP裂解酶首先进行克隆，在大肠杆菌中高效表达，然后进行纯化。用电子吸收、磁圆二色谱、电子顺磁共振、共振拉曼、穆斯堡尔谱等光谱技术对SP裂解酶的铁硫团簇进行了表征。将建立AM活性测定方法，探讨铁硫簇、S-腺苷蛋氨酸和还原剂对酶活性的影响。将使用两种不同的方法来确定SP裂解酶与DNA的结合，即DNA足迹和凝胶移位实验。将使用两种不同的方法来确定SP裂解酶与DNA的结合，即DNA足迹和凝胶移位实验。各种辅助因子的存在或不存在对DNA结合的影响也将通过这些方法进行检测。快速冷冻-淬火技术将用于光谱探测反应中间体，包括自由基中间体的可能性。最终，目标是了解SP裂解酶修复胸腺嘧啶二聚体的机制，包括铁-硫簇和腺苷甲酮的详细作用。

The specific aim of this proposal is to develop a mechanistic understanding of how spore photoproduct (SP) lyase, an adenosylmethione-dependent iron-sulfur enzyme, repairs DNA. SP lyase will first by cloned, over-expressed in E. coli, and then purified. The iron- sulfur cluster of SP lyase will be characterize with spectroscopic techniques including electronic absorption, magnetic circular dichroism, electron paramagnetic resonance, resonance Raman, Mossbaur spectroscopy. Am activity assay will be developed to probe the necessity for the iron-sulfur cluster, S-adenosylmethionine, and reducing agents on the enzyme activity. The binding of SP lyase to DNA will be determined using two different methods, DNA footprinting and the gel shift assay. The binding of SP lyase to DNA will be determined using two different methods, DNA footprinting and the gel shift assay. The effect on DNA binding of the presence or absence of various co-factors will also be examined by these methods. Rapid freeze-quench techniques will be used to spectroscopically probe reaction intermediates, including the possibility of radical intermediates. Ultimately, the goal is to gain an understanding of the mechanism by which SP lyase repairs thymine dimers, including the detailed roles of the iron-sulfur cluster and adenosylmethione.