Communication between protein and DNA is one of the most fundamental processes in all living cells. To understand the structural basis for this process in the Eschericia coli RI restriction-modification system, one needs to know the three-dimensional structures of all the players involved: they are the structures of free DNAs with and without the recognition sequence, the free endonuclease, the free methylase, the complexes between the endonuclease and DNA with and without the cognate sequence, and the corresponding complexes with the methylase. Of these eight structures only two, a free DNA containing the recognition sequence and a recognition complex, have been determined so far by X-ray crystallographic methods. Our overall objective is to determine the three-dimensional structures of the remaining six structures plus the recognition complexes with different flanking sequences. We would like to establish the structural basis for understanding the DNA sequence specific/non-specific interaction process and the dependence of the recognition on the flanking sequences at atomic resolution. We have constructed an over-producing strain of E. coli for the production of the endonuclease and methylase, from which we have prepared gram quantities of the enzymes. Several DNA sequences with and without the cognate sequence have also been synthesized. From these have been obtained single crystals of one DNA sequence, the endonuclease alone, and the complex between the endonuclease and a cognate DNA. X-ray crystallographic studies on these crystals and crystallization of the others are in progress.
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