Understanding DNA specificity in the IHF/HU family

描述（由申请人提供）：这些研究有两个总体目标：第一，进一步了解蛋白质-DNA相互作用，特别是DNA结构在位点识别中的作用，第二，更好地了解已选择用于详细研究的高度保守的HU/IHF蛋白质家族的生物学作用。蛋白质对特定DNA位点的识别是调控基因表达和许多其他生物过程的必要条件。在某些情况下，这种识别是正确排列的蛋白质侧链和B型DNA大沟中碱基边缘之间氢键的直接结果。然而，在许多情况下，情况要复杂得多，并且识别取决于DNA结构中的序列依赖性变化和扭曲性（“间接读出”）。这种形式的承认不像前者那样得到很好的理解。 IHF/HU家族蛋白是小的、密切相关的原核DNA弯曲蛋白，其在需要多组分蛋白-DNA复合物的多种过程（例如转录和重组）中作为结构因子起作用。两种待研究的蛋白质，IHF和Hbb，识别DNA中的特定（但不同）序列，但几乎完全通过间接读出。第三种是HU，几乎不依赖于DNA序列，但识别DNA中的特定结构扭曲，并可能在DNA修复中发挥作用。众所周知，IHF结合会在DNA中引入近180度的弯曲，但对HU引入的弯曲的估计差异很大。这种不确定性阻碍了我们对HU如何与细胞中的其他蛋白质一起发挥作用的理解。 该项目将研究这些蛋白质在溶液中和X射线晶体学中的结合特性。将通过定点诱变和结构域交换实验测试基于比较和对比待研究的3种不同蛋白质的此类数据的假设。

DESCRIPTION (provided by applicant): These studies have two overall goals: First, to further our understanding of protein-DNA interactions, particularly the role of DNA structure in site recognition, and second, to better understand the biological roles of the highly conserved HU/IHF family of proteins that have been selected for detailed study. Recognition of particular DNA sites by proteins is the sine qua non for the regulation of gene expression and many other biological processes. In some cases, such recognition is a straightforward consequence of hydrogen bonds between properly arrayed protein side chains and the edges of bases in the major groove of B-form DNA. In many instances, however, the situation is much more complicated, and recognition depends on sequence - dependent variations in the structure and distortability of the DNA ("indirect readout"). This form of recognition is not as well understood as the former. The IHF/HU family proteins are small, closely related prokaryotic DNA bending proteins that function as architectural factors in a variety of processes (e.g. transcription and recombination) that require multicomponent protein-DNA complexes. Two of the proteins to be studied, IHF and Hbb, recognize specific (yet different) sequences in the DNA, but do so almost entirely through indirect readout. The third, HU, binds nearly independently of DNA sequence but recognizes specific structural distortions in DNA, and may play a role in DNA repair. It is well established that IHF binding introduces a nearly 180 degree bend in the DNA, but estimates of the bend introduced by HU vary widely. This uncertainty hampers our understanding of how HU functions in conjunction with other proteins in the cell. This project will study the binding properties of these proteins both in solution and by x-ray crystallography. Hypotheses based on comparing and contrasting such data for the 3 different proteins to be studied will be tested by site-directed mutagenesis and domain-swap experiments.