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（由应用程序提供）：该赠款建议实验，以解决关键的病毒DNA包装计划和易位问题。噬菌体T4 DNA包装机制通过可比的运动蛋白（2）通过Prohead门户（2）广泛共享，其中大末端亚基（TERL）负责转运和小末端酶（TERL）和小型末端酶（TER）（TER）（TERS），用于包装的启动式contiative intiative intiative intiative of Concative intiative intiative。具体目标1将建立大的GP17（TERL）易位机制。我们提出了一种“ DNA Crunching”线性运动机制，该机制采用了B-形式DNA的握力和释放的瞬态弹簧压缩。我们的FRET测量值直接在体外包装停滞的Y-DNA底物中支撑此机制，该机制显示从末端酶到门户的距离降低；此外，在Y型茎DNA中，符合B-和A结构的近距离定位染料对之间的距离有所下降。在正常翻译中，TERL电动机排出无法结合A形式的所有B形式紧密结合的Yoyo-1染料。电动机无法包装A形式DSRNA或A形DNA：RNA异形电子化合物。我们的工作表明，添加了辅助B形DNA：DNA（D：D）20mers允许（D：R）杂质的A-Form DNA包装：RNA 20mers（D：R），B-TO A-A-Form Spring Motor的其他证据。此外，对荧光染料释放，光链染料的TERL交联和高分辨率结构数据的动力学分析将为A-A-A-Form运动机制提供支持和见解。 TERL结构域的晶体学和冷冻EM停靠在Prohead，门户和门户网站的夹子区域，将确认末端酶对接的C末端核酸酶结构域是到该门户网站的，如FRET和SDM分析所示。具体的目标2将确定噬菌体T4的小末端亚基GP16（TERS）在DNA PAC位点相互作用中以及通过双Ters环机构的包装倡议中的作用。 FRET测量和超分辨率显微镜将确认T4 TERS蛋白质以双环形式起作用以启动包装。功能性TERS-GFP和TERS-MCHERRY融合蛋白在体外和体内用作标准。 FRET的工作表明，TS突变体形式在低温但不是高温下形成环，表明函数需要形成环。仅在TERS蛋白质制剂中发现的双22mer和单个11mer环与DNA包装有何关系？强有力的遗传证据通过TERS的双环形式支持两个同源PAC DNA的突触，该双环形式反对四链PAC DNA结构，以霍利迪连接链交换DNA convatemer成熟来判断包装启动。