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H-1 HYPERSITES

H-1 超站点

基本信息

批准号：
2772082
负责人：
KENSAL E. VAN HOLDE
金额：
$ 2.44万
依托单位：
OREGON STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1996
资助国家：
美国
起止时间：
1996-09-01 至 2000-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2772082
关键词：
DNA binding protein DNA footprinting analytical ultracentrifugation gel electrophoresis gel mobility shift assay genome histones plasmids scanning tunneling microscopy

项目摘要

DESCRIPTION Recent findings suggest that the binding of the linker histone H1 to DNA may be more specific than previously believed, involving recognition of either certain nucleotide sequences or certain structural features of DNA (e.g. supercoils, 4-way junctions). The foreign collaborator recently discovered two plasmid restriction fragments that exhibit unusually high affinity for H1, based on results obtained with a simple gel shift electrophoresis assay. Preliminary studies of these H1 "hypersites" suggest that DNA curvature as well as specific sequences play a role in the high affinity binding. The proposed collaboration includes experiments which will test the hypothesis that the hypersite-containing DNA fragments form loops which place high-affinity sequences at or near a DNA cross-over. Initial experiments will verify the requirement for DNA curvature in H1-binding DNA fragments. Loop formation will be evaluated indirectly by gel electrophoresis and directly by scanning force microscopy. Two approaches will be used to determine if the binding of H1 to DNA hypersites is cooperative: first, by Western blotting to localize H1 in the gel shift assay, and second, by analytical ultracentrifugation. A third specific aim is to map the putative specific sequences involved in tight H1 binding; this will be attempted using a DNase protection assay. The final specific aim is to search for H1 hypersites in selected eukaryotic genome regions using the gel shift assay and to characterize these sites using the approach applied to the hypersite-containing plasmid fragments.

描述最近的研究结果表明，连接蛋白H1与DNA的结合可能比以前认为的更具体，包括承认 DNA的某些核苷酸序列或某些结构特征（例如，超螺旋、4路接头）。外国合作者最近发现两个质粒限制性片段显示出异常高的亲和力， H1，基于用简单凝胶移位电泳测定获得的结果。对这些H1“超位点”的初步研究表明，以及特异性序列在高亲和力结合中起作用。的拟议的合作包括将测试假设的实验，含有超位点的DNA片段形成环，在DNA交叉处或附近的高亲和力序列。最初的实验将验证DNA曲率的要求， H1结合DNA片段。将通过以下方式间接评价环形成：凝胶电泳和直接通过扫描力显微镜。两方法将用于确定H1与DNA超位点的结合是否是合作的：首先，通过Western印迹定位H1在凝胶移位第二，通过分析超离心。第三个具体目标是绘制与H1紧密结合有关的推定的特异性序列;这将尝试使用DNA酶保护测定。最终的具体目标是在选择的真核基因组区域中使用凝胶迁移试验，并使用所应用的方法表征这些位点含有超位点的质粒片段。