My goal is to determine, using NMR, the structure of a vaccinia virus DNA sequence containing two extrahelical bases (G and C) on the same strand, separated by d(AA).d(TT). It appears at each terminus of the genome, with the extrahelical bases at corresponding locations but as complements. I am testing alternative structural models by restrained molecular dynamics, using the progrm MARDIGRAS, based upon both NOE- derived proton-proton distances and relaxation matrix-calculated NOESY cross peak volumes in both D2O and H2O. I will particularly emphasize assessment of the extent of pairing of the two AT base pairs between the extrahelical bases and the looping out or stacking of the extrahelical bases themselves. The analysis of my experiments is heavily dependent upon the resources of the Computer Graphics Laboratory, both for the analytical software, including molecular dynamics, and for molecular modeling of the calculated structures using MidasPlus. My research is biologically significant in many respects. First, the mismatched sequence is required for resolution in the replication of poxvirus DNA, and the structure is needed for a molecular explanation. Second, the structure forms during the extrusion of the imperfect palindrome of the poxvirus replicative intermediate (cloned into a closed circular plasmid vector) to form the hairpin structure of the mature virus genome. Experimentally, this mismatched structure requires a considerable free energy to extend the cruciform. Finally, terminal hairpin loop structures are characteristic of the DNA of several eucaryotic viruses and organelles, including the iridoviruses, parvoviruses, phycodnaviruses and poxviruses; and the rDNA from Tetrahymena, the mitochondrial DNA of Paramecium and the linear mitochondrial DNA of yeast. Many of these terminal sequences contain extrahelical bases as bulges, loops or mismatches. Examples, in addition to vaccinia virus DNA, are the DNAs from the minute virus of mice, the Autographa californica multinucleocapsid nuclear polyhedrosis virus, the human parvovirus B19, the Leporipoxvirus Shope fibroma virus, and the linear mitochondrial DNA of yeast.
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