Functional Group Interactions in DNA - Protein Recognition

Boston College, Chestnut Hill MA

Investigators

Abstract

Abstract MCB-0077667 McLaughlin McLaughlin In order to probe the weak contacts between proteins and nucleic acids in sequence-specific complexes, isosteric nucleoside analogues will be employed to introduce incremental changes into the DNA recognition site, by what amounts to "atomic mutagenesis" of the functional group character. If a given functional group is critical to the formation of the protein-nucleic acid complex then an incremental response in the ability to form the complex should be present. If a functional group is unimportant, then no change in complex formation should occur. The most useful analogues are those that do not otherwise alter native inter-strand hydrogen bonding or other helix-stabilizing effects. The analogues used in this project will also permit the study of critical structural parameters such as the importance of the minor groove spine of hydration and/or metal ion binding for the stability of B-form DNA duplexes. Three sequence-specific protein-nucleic acid complexes will be examined during the course of this project: (i) The important transcriptional pre-initiation complex (TBP-TATA box complex), one that is critically important for eukaryotic transcription, will be studied. The complex itself is unusual in that the protein interacts with the A/T rich recognition sequence by binding solely through the minor groove. Newly developed minor groove base analogues will be used both to examine the importance of functional group interactions in the minor groove, as well as to determine the energetic cost of burying a hydrophilic functional group in a hydrophobic interface. (ii) A study of the Gene-4 proteins, responsible for the RNA primase activity in bacteriophage T7 will be pursued. Photo-affinity labeling techniques will be developed. The information generated should complement the data already obtained through nucleoside analogue studies. (iii) DNA polymerases and/or reverse transcriptases will be studied using analogues lacking the critical O2-carbonyl or N3-nitrogen in the minor groove. Differences between the DNA polymerase and reverse transcriptase (RT) activities may lead to a new class of RT chain terminators. The studies in this project 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.