Continued support is requested for the development of base and sequence specific spin-labeled nucleic acids, the analysis of their electron paramagnetic resonance (EPR) spectra by modern EPR theory, and their application as sensitive macromolecular probes with pmol to nmol of label. EPR spectra of oligonucleotide duplexes of different lengths labeled sequence specifically with 1-, 1-, and 2-atom tethered nitroxides will be analyzed by rigorous computational approaches to scrutinize motional models. Complexes between EcoRI endonuclease and duplex DNA 26mers, containing the EcoRI restriction site and strategically placed spin-labels, will be studied by EPR spectroscopy, and the EPR line shapes will be analyzed in conjunction with X-ray diffraction data and molecular modeling studies of spin-labeled EcoRI endonuclease - DNA complexes. This will allow us to determine whether potential changes in the EPR spectrum are the result of direct interactions between probe and protein or structural/dynamic changes of DNA at some distance from the DNA binding site. Gene 5 protein, complexes to (dT,L)36 in the """"""""oligonucleotide"""""""" or """"""""polynucleotide"""""""" binding mode, will be studied by EPR spectroscopy with the spin probe L located in well defined positions with respect to the 5' end of (dT,L)36. The EPR data, together with all available results from other techniques, will be used for molecular modeling of the gene 5 protein - DNA complex. With respect to the single-strand-binding protein from E. coli (SSB), the experiments will be focused on achieving a better mechanistic understanding of the advancing replication fork in prokaryotic systems. The question of whether interactions of pol III holoenzyme with SSB are present on ssDNA causing the release of SSB from the lattice will be tested with macromolecular probes containing pause signals.
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