PROTEINS IN THE GAMMA DELTA RESOLVASE SYNAPTOSOME

六个二聚体的γ-δ解离酶和两个拷贝的120碱基对DNA 节（res）形成一个特定的蛋白质-DNA复合物，突触体，在 位点特异性重组。Resolvase二聚体与三个类似的 每个保留地内的地点（地点一、二和三）;然而， 解离酶是不同的，因为这些位点和DNA切割和连接发生 仅在站点I。这表明，在站点I和 突触体内的位点II和III在重要方面不同。一个 关于解离酶反应的一个重要且未回答的问题 涉及两个交叉位点的突触。两个人是怎么解决的-- 在突触复合体中，位点I的结合拷贝彼此相互作用 从而促进链断裂和交换？的x射线晶体结构 结合到站点I的解离酶提供了关于这种相互作用的一些线索 到目前为止，诱变还没有鉴定出相关的蛋白质 接口. 研究突触内I位点的解离酶的排列， 复杂的，有针对性的蛋白质足迹将被用作一种工具， 测定解离酶二聚体结合的蛋白-蛋白相互作用， 站点I.与DNA共价结合的解离酶的分离 位点I（通过丝氨酸-10-a 5'磷酸）将用于区分 解离酶分子在站点I从那些在站点II和II从自由 分解酶表面氨基酸残基的化学修饰和 用特异性蛋白酶切割将有助于鉴定溶剂改性 表面氨基酸残基和切割与特定的蛋白酶将 有助于鉴定溶剂可及和埋藏的氨基酸残基 突触复合体中的解离酶。解离酶的定点突变 参与突触复合体作用的表面残基 大分子的相互作用将被用来证实它们在 I位点的解离酶作用。

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.