INTERACTIONS OF P53 AND HMG-I(Y) IN SPERMATOGENESIS

该提案有两个主要目标：(1)研究和阐明P53和人HMG-I(Y)在合成连接DNA上的特异性相互作用及其相互作用的可能性；(2)研究P53启动子的染色质结构以及HMG-I(Y)结合对染色质结构的影响。第一个步骤包括确定这些蛋白质与四向连接的结合是否相互排斥，以及在碱基对分辨时确定这些蛋白质的结合位点。第二项研究将检测HMG-I(Y)在体外和体内对P53启动子染色质结构的影响。此外，将通过化学交联研究和抗体检测方法确定特定的HMG-I(Y)-P53蛋白质-蛋白质相互作用。合成的四向连接、P53共识位点和P53启动子区域将被用作DNA底物。这些实验将包括羟基自由基足迹、电泳迁移率、报告基因表达和光交联技术。

The proposal has two broad goals: (1) to study and elucidate the specific interactions of p53 and human HMG-I(Y) on synthetic junction DNA and the possibility that the proteins interact; and (2) to study the chromatin structure of the p53 promoter in vitro and in vivo and the effects of HMG-I(Y) binding on chromatin structure. The first includes determining whether binding of these proteins to four-way junctions is mutually exclusive and identifying the binding sites of these proteins at base-pair resolution. The second will examine the effect of HMG-I(Y) on the p53 promoter chromatin structure in vitro and in vivo. Additionally, specific HMG-I(Y)-p53 protein-protein interactions will be identified by chemical crosslinking studies and antibody detection methods. Synthetic four-way junctions, p53 consensus sites, and the p53 promoter region will be used as DNA substrates. These experiments will include hydroxyl radical footprinting, electrophoretic mobility, reporter gene expression, and photocrosslinking techniques.