We propose to use the highly specific interaction between EcoRI endonuclease and oligonucleotides containing the sequence d(GAATTC) as a model system for the study of sequence specific DNA-protein recognition. The principal goals of this research are to dissect the overall function of the endonuclease into operationally defined component activities; and then to use these data to develop a detailed functional description of the interaction between this restriction enzyme and its cognate DNA. We have shown that this functional dissection can be achieved through the use of mutationally or proteolytically altered forms of the enzyme. Here we propose to build on this foundation by investigating four specific areas: a) to identify amino acid residues which are essential for binding function by chemical modification techniques; b) to identify, by chemical modification, the regions of the molecule which participate in conformational mobility upon DNA binding; c) to ask whether or not a tryptic core endonuclease which has lost DNA cleavage ability retains the full sequence specificity of intact enzyme and also whether or not the electrostatic component of the DNA-protein interaction has been altered; d) to identify DNA phosphate contacts made by a mutant endonuclease having decreased electrostatic interaction with DNA.
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