NMR STUDIES OF VACCINIA VIRUS DNA SEQUENCES

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The DNA of vaccinia virus is a large (180 kbp) linear duplex with cross-linked termini, such that a single strand circle forms upon denaturation. The terminal 10 kbp are inverted repeats, and the ultimate 106 nucleotides at each end form incompletely base paired hairpins; they are involved in the formation of replicative intermediates of vaccinia virus. The terminal hairpins are extremely AT-rich (96% AT) and contain an asymmetric internal loop and a number of single base bulges. We are interested in a structure of a vaccinia virus DNA fragment which contains two unpaired residues (G and C) within the context of a 100% AT-rich duplex. We designed a construct containing this sequence (a 29-mer hairpin loop DNA) and a corresponding control construct lacking the unpaired residues (a 27-mer hairpin loop DNA), and acquired sets of 2D proton NMR data for high-resolution structure calculations. We have nearly completed proton resonance assignments for both molecules, and currently we are carrying out structure calculations for the control 27-mer sequence. The interactive computer graphics software developed at the Computer Graphics Laboratory is used at all stages of the refinement: firstly, to examine the self-consistency of the experimental restraints, then, to monitor the course of the refinement, and finally, to visualize and analyze the final structure.