PROTEINS IN THE GAMMA DELTA RESOLVASE SYNAPTOSOME

六个伽马三角洲分解酶二聚体和两个拷贝的120碱基对DNA 片段(RES)形成一种特定的蛋白质-DNA复合体，即突触体 特定部位重组。溶血酶二聚体与三个类似的 每个RES内的站点(站点I、站点II和站点III)；然而， 随着这些位点和DNA裂解和连接的发生，分解酶是不同的 仅在站点I。这表明站点I和站点I的解析酶相互作用 突触体内的II和III位点在重要方面有所不同。一个 关于分解酶反应的重要和未回答的问题 关于两个交叉点的突触。如何解决这两个问题- 位点I的结合拷贝在突触复合体中相互作用 从而促进链切割和交换？X射线晶体结构 绑定到站点I的解析器提供的关于此交互的线索很少 到目前为止，诱变还没有鉴定出相关的蛋白质。 界面。 研究突触中I点的分解酶的排列 复杂的、有针对性的蛋白质足印将被用作 测定结合于的解旋酶二聚体的蛋白质-蛋白质相互作用 位点I.与DNA ATE结合的共价结合解旋酶的分离 位点I(通过丝氨酸-10-α5‘磷酸)将用于区分 第一位点的解旋酶分子来自第二位点和第二位点的游离分子 解析器。表面氨基酸残基的化学修饰和 特定蛋白水解酶的切割将有助于识别溶剂修饰 表面氨基酸残基和特定蛋白酶的裂解将 帮助确定溶剂可及和埋藏的氨基酸残基 突触复合体中的分解酶。解旋酶的定点诱变 表面残基在突触复合体中的作用 将使用大分子相互作用来确认它们在 站点I中的解析操作。

Six dimers of gamma delta resolvase and two copies of a 120 base pair DNA segment (res) form a specific protein-DNA complex, the synaptosome, during site-specific recombination. Resolvase dimers are bound to three similar sites within each res (sites I, II and III); however, the role of resolvase is different as these sites and DNA cleavage and ligation occurs solely at site I. This suggests that resolvase interaction at site I and sites II and III within the synaptosome differ in important ways. An important and unanswered question regarding the resolvase reaction concerns synapsis of the two crossover sites. How do the two resolvase- bound copies of site I interact with one another in the synaptic complex so as to promote strand cleavage and exchange? The X-ray crystal structure of resolvase bound to site I provides few clues regarding this interaction and, thus far, mutagenesis has failed to identify the relevant protein interface. To investigate the arrangement of resolvase at site I in the synaptic complex, targeted protein foot printing will be used as a tool for determining the protein-protein interaction of resolvase dimers bound at site I. The isolation of covalently attached resolvase bound to DNA ate site I (via serine-10-a 5' phosphate) will be used to distinguish resolvase molecules at site I from those at site II and II from free resolvase. Chemical modification of surface amino acid residues and cleavage with specific proteases will help identify solvent modification of surface amino acid residues and cleavage with specific proteases will help identify solvent accessible and buried amino acid residues of resolvase in the synaptic complex. Site-directed mutagenesis of resolvase surface residues implicated in having a role in synaptic complex macromolecular interactions will be used to confirm their importance in resolvase action in site I.