During the morphogenesis of all studied double-stranded DNA bacteriophages, a DNA-free protein capsid is assembled and this capsid subsequently packages the bacteriophage's DNA The bacteriophages studied here cut their mature DNA from a longer (concatemeric) DNA. The long-term objectives of the research proposed are to determine, the pathway of DNA packaging, the sources of energy for packaging, the sources of information for packaging and the constraints on packaging during evolution.
The specific aims are to: (a) determine the free energy of DNA packaging by use of the Clausius-Clapeyron equation; measurement of packaged DNA's expulsion kinetics as a function of colloid osmotic pressure and temperature will be made by use of fluorescent dye binding assays; (b) determine the arrangement of DNA packaged in mature and immature capsids; quantitation of structure probe (including several intercalators) binding and analysis of chemically-induced cross linking will be performed by use of quantitative fluorimetry, electron microscopy and ge electrophoresis; (c) determine of the states achieved by the capsid during DNA packaging electron microscopy, immuno-electron microscopy, quantitative agarose gel electrophoresis binding of fluorescent structure probes, protein-protein crosslinking, sodium dodecyl sulfate polyacrylamide gel electrophoresis and protease digestion will be used; (d) isolate new DNA packaging intermediates; packaging will be slowed and intermediates stabilized by use of intercalators and bacteriophage mutations, the latter obtained by high intensity gene-specific mutagenesis; intermediates will be isolated from either infected cells or from our recently-developed high efficiency """"""""in vitro"""""""" DNA packaging extracts; (e)improve understanding of the structure of concatemers and other forms of DNA present during DNA packaging; DNA will be characterized by electron microscopy, gel electrophoresis (including rotating gel electrophoresis, developed by the PI), restriction endonuclease digestion and DNA-DNA hybridization; (f) increase the capabilities of gel electrophoresis for isolating and characterizing crosslinked, branched, single-stranded and long (more than 40 Kb) duplex DNA. The above studies will be conducted primarily with bacteriophage T7. To help determine evolutionary constraints on packaging mechanisms, some studies will also be done with bacteriophages lambda, P22, T3 and T4. Data to be obtained have potential use in antiviral therapy; an example is quantitation of completely and partially packaged DNA's binding to intercalators. Techniques to be improved have several biotechnological and biomedical applications, including the mapping of eukaryotic DNAs.
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