The goal of the proposed research is to develop chemical and enzymatic approaches for the study of nucleic acid structure and nucleic acid-protein interactions. The present studies focus on the interactions of the restriction and modification enzymes with the specific DNA sequences. Recently, we have isolated four restriction and modification enzymes frm Haemophilus parainfluenza in homogeneous form and have studied their catalytic properties. Extending these studies, we propose to investigate the functional groups of both protein(s) and DNA(s) which interact with each other to establish the highly developed specificity of these enzymes. Specifically we propose to: (a) determine all the nucleotide functional groups which interact with the enzyme. We have already established two contact points between the recognition sequence, dGTTAAC and Hpa I endonuclease. We will seek to determine other contact points by replacing other nucleotide bases with their base analogs. In addition, we will extend our previous studies to determine as to how the surrounding sequences play a role in establishing precise interaction of protein and the recognition sequence. (b) Localize the amino acids or functional groups of the enzyme which interact with the DNA sequence. These studies require the knowledge of the amino acid sequence of the enzyme from the cloned gene sequence. From the knowledge of the primary sequence of the enzyme, we can begin to design chemical modifications of the selective amino acids which may be involved in the interaction of the DNA. Most of these studies utilize the techniques developed in this laboratory in the last three years.
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