NMR STUDIES OF VACCINIA VIRUS DNA SEQUENCES

痘苗病毒 DNA 序列的 NMR 研究

• 批准号: 6456813
• 负责人: NIKOLAI B ULYANOV
• 金额: $ 27.32万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2003-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6456813
• 关键词: bacteria; biomedical resource; environmental toxicology; enzymes

The DNA of vaccinia virus is a large (180 kbp) linear duplex with cross-linked termini, such that a single strand circle forms upon denaturation. The terminal 10 kbp are inverted repeats, and the ultimate 106 nucleotides at each end form incompletely base paired hairpins; they are involved in the formation of replicative intermediates of vaccinia virus. The terminal hairpins are extremely AT-rich (96% AT) and contain an asymmetric internal loop and a number of single base bulges. We are interested in a structure of a vaccinia virus DNA fragment which contains two unpaired residues (G and C) within the context of a 100% AT-rich duplex. We designed a construct containing this sequence (a 29-mer hairpin loop DNA) and a corresponding control construct lacking the unpaired residues (a 27-mer hairpin loop DNA), and acquired sets of 2D proton NMR data for high-resolution structure calculations. We have nearly completed proton resonance assignments for both molecules, and currently we are carrying out structure calculations for the control 27-mer sequence. The interactive computer graphics software developed at the Computer Graphics Laboratory is used at all stages of the refinement: firstly, to examine the self-consistency of the experimental restraints, then, to monitor the course of the refinement, and finally, to visualize and analyze the final structure.

痘苗病毒的 DNA 是一个大的 (180 kbp) 线性双链体， 交联末端，使得单链环形成 变性。 末端 10 kbp 是反向重复序列， 每端最终 106 个核苷酸形成不完全碱基配对 发夹；它们参与复制的形成 痘苗病毒的中间体。 末端发夹非常 富含 AT（96% AT）并包含不对称内环和多个 单底座凸起。 我们对牛痘的结构感兴趣 包含两个不配对残基（G 和 C）的病毒 DNA 片段 在 100% 富含 AT 的双链体中。 我们设计了一个结构 包含该序列（29 聚体发夹环 DNA）和 缺少未配对残基的相应对照构建体（a 27 聚体发夹环 DNA），并获取了 2D 质子 NMR 数据集 高分辨率结构计算。 我们已经快完成了 两个分子的质子共振分配，目前我们正在 对对照 27 聚体序列进行结构计算。 计算机学院开发的交互式计算机图形软件 图形实验室用于细化的所有阶段：首先， 检查实验限制的自我一致性，然后， 监控细化过程，最后可视化和 分析最终的结构。