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: 9080621
• 负责人: LINDSAY W BLACK
• 金额: $ 30.42万
• 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/9080621
• 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; Figs - dietary; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Genetic; Goals; Grant; Health; Herpesviridae; High temperature of physical object; Human; In Vitro; 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; Twin Multiple Birth; United States National Institutes of Health; Virus Assembly; Work; base; cold temperature; crosslink; ds-DNA; grasp; in vivo; insight; mutant; novel; nuclease; public health relevance; research study; 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(由申请者提供)：这项资助建议进行实验，以解决关键的病毒DNA包装、启动和移位问题。噬菌体T4 DNA包装机制在可比噬菌体中广泛共享，这些噬菌体通过可比的马达蛋白(2)通过前头门户包装dsDNA，其中大末端酶亚单位(TerL)负责转位，小末端酶(TERS)负责串联体的包装起始切割。具体目标1将建立大gp17(TerL)易位机制。我们提出了一种“DNA碾压”直线电机机构，它采用了一种抓取和释放B-型到A-型DNA的瞬时弹簧式压缩。我们的FRET测量直接支持这一机制，在体外包装停滞的Y-DNA底物，显示从终止酶到门户的距离减少；此外，Y-茎DNA中紧密定位的染料对之间的距离减少，符合B-和A-结构。在正常的移位中，TerL马达会排出所有B型的紧密结合的YOYO-1染料，而这些染料不能结合A型。马达不能包装A型dsRNA或A型DNA：RNA异源双链。我们的工作表明，辅助B型DNA：DNA(D：D)20mer的加入允许(D：R)包装异质双链A型DNA：RNA20mer(D：R)，这是B-to A型弹簧马达的额外证据。此外，荧光染料释放的动力学分析，光可连接染料的TerL交联，以及高分辨率结构数据将为这一拟议的B-型到A-型马达机制提供支持和洞察。如FRET和SDM分析所示，将TerL结构域对接到探针、门户和门户的剪切区的结晶学和冷冻EM将证实末端酶的C-末端核酸酶结构域与门户对接。具体目标2将确定噬菌体T4的小末端酶亚单位gp16(TERS)在DNA PAC位点相互作用和通过双TERS环机制启动包装中的作用。FRET测量和超分辨率显微镜将证实T4TERS蛋白以双环形式启动包装。以体内外功能性TERS-GFP和TERS-mCherry融合蛋白为标准。FRET研究表明，TERS蛋白的TS突变形式在低温度而不是高温下形成环，表明功能需要环形成。在仅含蛋白质的TERS制剂中发现的双22聚体和单11聚体环与DNA包装有何关系？强有力的遗传学证据支持两个同源的PAC DNA通过与四链PAC DNA结构相反的TERS的双环形式突触，以Holliday结链交换DNA链成熟来判断包装的起始。