Bacteriophage phi29 of Bacillus subtilis is a small, well characterized virus containing a double-stranded DNA of 18 Kbp. Its entire genome has been sequenced and each of twenty-three genes is marked with suppressor-sensitive (sus) or temperature sensitive (ts) mutants. The products of most of the genes have been identified, and structural proteins are assembled in a single morphogenetic pathway. We have described a completely defined in vitro assembly system in which the phi29 DNA with covalently bound gp3 is packaged into a prohead with the aid of the single protein gp16 (PNAS, 83, 3505, 1986) and with an efficiency equivalent to in vivo assembly. The present proposal is centered on an analysis of the topology of packaged DNA-gp3, and the structure and function of the DNA packaging protein gp16, a DNA-dependent ATPase that topologically modifies phi29 DNA- gp3 as a prerequisite for packaging. We will study the intravirion topology of DNA-gp3 by ion etching, which progressively erodes virion components from the outside to the inside while preserving the overall structure. By use of this method and virions assembled in vitro, we will determine 1) the location of the left and right ends of the genome in the capsid; 2) the location of gp3; and 3) the presence of sharp (180 degrees) bends in the parallel DNA strands. We will study the binding of the DNA-gp3 packaging protein gp16 to DNA-gp3 that induces topological modification of the DNA during in vitro assembly. We will study the stoichiometry and specificity of binding of gp16 to DNA-gp3. Interaction of gp16 with specific segments of DNA will be studied by footprinting or by use of methylation. Negative superhelical density in DNA/gp3-gp16 complexes will be measured by the use of psoralen photoaffinity probes and the rate of photobinding. We will seek evidence for formation of nucleosome-like structures with an altered DNA helical pitch by the use of nuclease digestion experiments. The molecular structure of gp16 will be studied. We will attempt to identify the functional domains of gp16 involved in prohead, DNA-gp3, and ATP interactions. We will perturb local structures of the protein by cloning altered DNA and characterizing genetically altered proteins for function in DNA binding and topological modification. We will attempt to produce 2-D and 3-D crystals of gp16; 2-D crystals will be analyzed by Fourier filtering of electron micrographs and 3-D crystals will be analyzed by X-ray diffraction.
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