Mechanism of bacteriophage DNA packaging initiation and DNA translocation.

噬菌体DNA包装起始和DNA易位的机制。

• 批准号: 9274832
• 负责人: LINDSAY W BLACK
• 金额: $ 30.51万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-01 至 2020-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9274832
• 关键词: A-Form DNA; ATP Hydrolysis; ATPase Domain; Adenoviruses; Antiviral Agents; B-DNA; Bacteriophage T4; Bacteriophages; Binding; Biological Models; Bromides; C-terminal; Chimeric Proteins; Chromosome Pairing; Clip; Complex; Concatenated DNA; Cruciform DNA; Crystallography; DNA; DNA Packaging; DNA Structure; Data; Docking; Double-Stranded RNA; Dyes; Ethidium; Excision; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Genetic; Goals; Grant; Health; Human; In Vitro; Influenza; Intervention; Kinetics; Laboratories; Length; Linear Models; Mass Spectrum Analysis; Measurement; Medical; Microscopy; Mission; Modeling; Motor; Mutation; N-terminal; Optics; Peptides; Positioning Attribute; Preparation; Process; Proteins; RNA; Resistance; Resolution; Role; Signal Transduction; Site; Solid; Structure; Torsion; Twin Multiple Birth; United States National Institutes of Health; Virus Assembly; Work; base; cold temperature; crosslink; ds-DNA; experimental study; grasp; in vivo; insight; mutant; novel; nuclease; public health relevance; stem; terminase; viral DNA

 DESCRIPTION (provided by applicant): This grant proposes experiments to resolve critical viral DNA packaging initiation and translocation issues. Bacteriophage T4 DNA packaging mechanisms are widely shared among comparable phages packed with dsDNA through a prohead portal by comparable motor proteins (2), where the large terminase subunit (TerL) is responsible for translocation and the small terminase (TerS) for packaging initiation-cutting of the concatemer. Specific Aim 1 will establish the large gp17 (TerL) translocation mechanism. We propose a "DNA crunching" linear motor mechanism that employs a grip-and-release transient spring-like compression of B- to A-form- DNA. Our FRET measurements directly support this mechanism in a packaging stalled Y-DNA substrate in vitro that show a decrease in distance from terminase to portal; furthermore, there is a decrease in distance between closely positioned dye pairs in the Y-stem DNA that conforms to B- and A- structure. In normal translocation the TerL motor expels all B-form tightly binding YOYO-1 dye that cannot bind A-form. The motor cannot package A-form dsRNA or A-form DNA:RNA heteroduplexes. Our work shows that addition of helper B- form DNA:DNA (D:D) 20mers allows (D:R) packaging of heteroduplex A-form DNA:RNA 20mers (D:R), additional evidence for a B- to A-form spring motor. Additionally, kinetic analyses of fluorescent dye release, TerL cross-linking of photo-linkable dye, and high resolution structural data will provide support and insight into this proposed B-form to A-form motor mechanism. Crystallography and cryo-EM of TerL domains docked to proheads, portals, and to a clip region of the portal will confirm that the C-terminal nuclease domain of the terminase docks to the portal, as shown by FRET and SDM analysis. Specific Aim 2 will establish the role of the small terminase subunit gp16 (TerS) of phage T4 in DNA pac site interaction and in packaging initiation by a twin TerS ring mechanism. FRET measurements and superresolution microscopy will confirm that the T4 TerS protein acts in a double ring form to initiate packaging. Functional TerS-GFP and TerS-mCherry fusion proteins in vitro and in vivo serve as standards. FRET work shows that a ts mutant form of the TerS protein forms rings at low but not high temperature, showing ring formation is required for function. How do the double 22mer and single 11mer rings found in TerS protein-only preparations relate to DNA packaging? Strong genetic evidence supports synapsis of two homologous pac DNAs by a twin ring form of the TerS that opposes a four stranded pac DNA structure to judge by Holliday junction strand swapping DNA concatemer maturation for packaging initiation.

 描述（由申请人提供）：这项资助提出了解决关键病毒 DNA 包装启动和易位问题的实验，噬菌体 T4 DNA 包装机制在通过类似马达蛋白通过前头门户包装的类似噬菌体中广泛共享 (2)，其中大终止酶亚基 (TerL) 负责易位，小终止酶 (TerS) 负责包装起始切割多联体，从而建立大的特定目标 1。 gp17 (TerL) 易位机制我们提出了一种“DNA 压缩”线性电机机制，该机制采用 B 型至 A 型 DNA 的夹紧和释放瞬态弹簧压缩，我们的 FRET 测量直接支持包装中的这种机制。体外 Y-DNA 底物停滞，显示从终止酶到门户的距离减小；此外，在正常易位中符合 B- 和 A- 结构的 Y-茎 DNA 中紧密定位的染料对之间的距离也减小。这TerL 马达排出所有不能结合 A 型的 B 型紧密结合的 YOYO-1 染料，该马达无法包装 A 型 dsRNA 或 A 型 DNA:RNA 异源双链体。我们的工作表明，添加辅助 B 型 DNA:DNA。 (D:D) 20mers 允许 (D:R) 包装异源双链 A 型 DNA:RNA 20mers (D:R)，另外还有 B 型到 A 型弹簧马达的额外证据，以及动力学分析。荧光染料释放、可光连接染料的 TerL 交联以及高分辨率结构数据将为该提议的 B 型到 A 型运动机制提供支持和深入了解与前体对接的 TerL 结构域的晶体学和冷冻电镜，门户，并到门户的剪辑区域将确认终止酶的 C 末端核酸酶结构域对接至门户，如 FRET 和 SDM 分析所示，将确定小端口的作用。噬菌体 T4 的终止酶亚基 gp16 (TerS) 在 DNA pac 位点相互作用和双 TerS 环机制的包装启动中，FRET 测量和超分辨率显微镜将证实 T4 TerS 蛋白以双环形式起作用以启动功能性 TerS-。体外和体内的 GFP 和 TerS-mCherry 融合蛋白作为标准 FRET 工作表明，TerS 蛋白的 ts 突变形式在低浓度但不高浓度下形成环。温度，表明在仅 TerS 蛋白制剂中发现的双 22mer 和单 11mer 环如何与 DNA 包装相关？强有力的遗传证据支持两个同源 pac DNA 通过 TerS 的双环形式进行联会。反对通过霍利迪连接链交换 DNA 串联体成熟来判断包装起始的四链 pac DNA 结构。