ULTRA HIGH RESOLUTION DIFFRACTION STUDIES OF FEN-1 AND OTHER DNA REPLICATION

FEN-1 和其他 DNA 复制的超高分辨率衍射研究

• 批准号: 7601573
• 负责人: LEIF HANSON
• 金额: $ 0.83万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7601573
• 关键词: Aeropyrum; Bacteriophage T4; Benchmarking; Binding; Complex; Computer Retrieval of Information on Scientific Projects Database; Condition; DNA; DNA biosynthesis; Data; Elements; Funding; Grant; Housing; Institution; Macromolecular Complexes; Measurement; Modeling; Molecular Machines; Neutron Diffraction; Neutrons; Pathway interactions; Protein Binding; Proteins; RTH-1 Nuclease; Research; Research Personnel; Resolution; Resources; Ribonuclease H; Roentgen Rays; SS DNA BP; Solutions; Source; Structural Models; Structure; Surgical Flaps; Techniques; United States National Institutes of Health; base; beamline; cell dimension; cold temperature; interest; multidisciplinary; repaired; research study; spleen exonuclease; ultra high resolution

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The proposed diffraction studies seek to obtain higher resolution data from proteins in the DNA replication pathway. These proteins are of interest in modeling and understanding the macromolecular complexes that function in DNA replication and repair. Multidisciplinary structural studies of the complexes (protein:DNA and protein:protein) and their constituents are underway using diffraction and scattering techniques with X-rays and neutrons. However the bases of all our studies are maximal resolution X-ray structural models of the protein elements that comprise these molecular machines. Of primary interest for this proposal are the proteins FEN-1, and the elements and ternary complex of RNase H:gp32-N:DNA. We seek ultra-high resolution diffraction data (<0.9A) of flap endonuclease 1 (FEN-1) protein from the Aeropyrum pernix. This extremophile protein that binds flap lagging strand DNA during replication has diffracted to 1.4¿ in previous experiments. However larger crystals combined with flash-cooling conditions that better preserve crystalline order have resulted in measurement of much higher resolution diffraction data in-house than has been seen previously. This protein is also the subject of low temperature neutron diffraction studies, both with H/D exchanged and perdeuterated proteins. Combined with different cooling regimes during diffraction, the protein will provide a useful benchmark for comparison of different data measurement techniques. The 5¿ to 3¿ exonuclease (RNase H) and the single-stranded DNA binding protein (gp32) from bacteriophage T4 form a transient complex during DNA replication. The X-ray structures of T4 RNase H and of the core of T4 gp32 are known. In solution, gp32 missing the N-terminus binds to RNase H and forms a stable complex in the absence of DNA. Crystal diffraction extends to 3.5¿ with a long cell dimension. High flux beamline data are essential to resolving the structures of this complex.

该副本是使用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这是调查员的机构。 拟议的衍射研究旨在从DNA复制途径中的蛋白质获得更高的分辨率数据。这些蛋白质在建模和理解在DNA复制和修复中起作用的大分子复合物方面具有兴趣。复合物的多学科结构研究（蛋白质：DNA和蛋白质：蛋白质）及其结构研究及其构造使用X射线和中子的衍射和散射技术正在进行中。但是，我们所有研究的基础是构成这些分子机器的蛋白质元件的最大分辨率X射线结构模型。该建议的主要感兴趣是蛋白质Fen-1，RNase H：GP32-N：DNA的元素和三元复合物。 我们寻求皮瓣核酸内切酶1（FEN-1）的超高分辨率衍射数据（<0.9a） 来自Aeropyrum Pernix的蛋白质。在复制过程中结合瓣滞后DNA的这种极端蛋白在先前的实验中衍射为1.4。但是，与以前相比，与以前相比，内部内部更高的分辨率衍射数据相比，较大的晶体结合了更好地保留晶体顺序的闪光冷却条件。该蛋白也是低温中子衍射研究的主题，无论是H/D提供了H/D的蛋白质。结合衍射过程中不同的冷却方案，该蛋白质将为比较不同数据测量技术的比较提供有用的基准。 从细菌噬菌体T4发出的5¿到3核（RNase H）和单链DNA结合蛋白（GP32）在DNA复制过程中形成了瞬态复合物。 T4 RNase H和T4 GP32核心的X射线结构已知。在溶液中，GP32缺少N末端与RNase H结合并在没有DNA的情况下形成稳定的复合物。晶体衍射延伸至3.5？，具有长细胞尺寸。高通量光束线数据对于解决该复合物的结构至关重要。