Research Project
3438594
A fundamental understanding of protein interaction with DNA at the molecular level may be a key in elucidating many critical steps in genetic phenomena. DNA restriction endonucleases and methylases are important enzymes for protection from the incorporation of foreign DNA in a host specific manner. In addition to their functional importance, however, these enzymes may serve as models for studying the site-specific interaction of proteins with DNA, and are of utility as tools for the molecular biologists. This proposal seeks to gain information regarding the interaction of restriction endonucleases and methylases with DNA as well as the mechanism of endonuclease action by studying the influence of phosphorothioate modification (substitution for phosphate) of DNA substrate on the enzymes. Structural information can be gained by studying these influences as a function of position and stereochemistry of the modification. Inhibition of endonucleases by the phosphorothioate will have implications on the mechanism of the action of the endonucleases. Additionally, a portion of this proposal is specifically directed towards enzymes that recognize an asymmetric sequence in the DNA, that is a sequence which is not identical to its complement (not totally palindromic). As an investigation designed to fulfill the criteria of the NIH Academic Research Enhancement Award (AREA) Program, these preliminary investigations should hopefully provide direction in focusing on a thorough elucidation of the structure-function relationship in the recognition of a specific DNA sequence by proteins and/or the mechanism of action of the enzymes.
