9723844 McLaughlin Recognition events involving enzymes, proteins or even simple ligands and sequences of single or double-stranded DNA are, in the simplest analysis, the result of specific ionic, hydrogen bonding and hydrophobic interactions present at the macromolecular interface. This research will determine the nature and the contribution of such intermolecular interactions to the observed high-affinity binding in such complexes by probing individual functional group contacts. Isosteric nucleoside analogues will be employed to introduce incremental changes in functional group character by what amounts to "atomic mutagenesis", and the effects of such incremental changes upon binding affinity, catalytic activity or simple structural parameters will be determined.. The analogues involve the "deletion modification" of a native nucleoside in which a specific functional group - amino, carbonyl, imino or methyl - is excised and replaced with a hydrogen atom. The most useful analogues are those which do not otherwise alter native inter-strand hydrogen bonding or other helix-stabilizing effects. This research will expand the current repertoire of analogues available to probe macromolecular recognition processes by developing analogues to probe contacts to functional groups present in the minor groove of a B-form DNA duplex as well as those to the internucleotide phosphodiester residues. These analogues also will permit the study of critical structural parameters such as the importance of the minor groove spine of hydration for the stability of B-form DNA duplexes, and the relation of this spine of hydration to the DNA curvature effect present in oligo A-T sequences. Three sequence-specific protein-nucleic acid complexes will be examined during the course of this project. The important eukaryotic transcriptional pre-initiation TBP-TATA box complex in which protein interacts with the A/T rich recognition sequence by binding solely through the DNA minor groove will be investigated using newly developed minor groove base analogues. The Gene-4 proteins responsible for the RNA primase activity in bacteriophage T7 will be examined with respect to the nature of recognition and catalysis by the T7 primase enzyme at the CTG recognition site on single-stranded DNA, as will the functional group requirements on the conserved but non-transcribed C residue and the transcribed T and G residues of the template. A new study of the integrase protein of ( bacteriophage will begin with a probe of the mechanistic details using 5'-phosphorothioate linkages as suicide substrates for the recombinase enzyme. These studies will help to further the understanding of DNA structure as well as sequence-specific protein-nucleic acid and ligand-nucleic acid interactions by elucidating the location and relative importance of functional group interactions that contribute to overall specificity and affinity in such complexes. %%% Recognition by enzymes, proteins and even simple molecules of (ligands) sequences of single or double-stranded DNA are, in the simplest analysis, the result of specific ionic, hydrogen bonding and hydrophobic interactions. This research will help to further the understanding of DNA structure as well as sequence-specific protein-nucleic acid and ligand-nucleic acid interactions by elucidating the location and relative importance of functional (atomic) group interactions that contribute to overall specificity and affinity in such complexes. ***
