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.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 提出的衍射研究寻求从DNA复制途径中的蛋白质获得更高分辨率的数据。 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重要意义。复合物（蛋白质：DNA和蛋白质：蛋白质）及其成分的多学科结构研究正在进行中，使用X射线和中子的衍射和散射技术。然而，我们所有研究的基础是组成这些分子机器的蛋白质元素的最大分辨率X射线结构模型。该提议的主要兴趣是蛋白质FEN-1，以及RNase H：gp 32-N：DNA的元件和三元复合物。 我们寻求瓣状核酸内切酶1（FEN-1）的超高分辨率衍射数据（<0.9A） 来自Aeropyrum pernix的蛋白质。这种在复制过程中结合皮瓣滞后链DNA的极端蛋白质在以前的实验中衍射到1.4 º。然而，更大的晶体与更好地保持晶体有序的快速冷却条件相结合，导致内部测量的衍射数据分辨率比以前高得多。这种蛋白质也是低温中子衍射研究的主题，包括H/D交换和全氘代蛋白质。结合不同的冷却制度在衍射过程中，蛋白质将提供一个有用的基准比较不同的数据测量技术。 第五章到3分核酸外切酶（RNase H）和来自噬菌体T4的单链DNA结合蛋白（gp 32）在DNA复制期间形成瞬时复合物。T4 RNase H和T4 gp 32核心的X射线结构是已知的。在溶液中，缺失N-末端的gp 32与RNase H结合，并在不存在DNA的情况下形成稳定的复合物。晶体衍射延伸至3.5 º，具有长晶胞尺寸。高通量束线数据是必不可少的，以解决这个复杂的结构。