CRYSTALLOGRAPHIC STUDIES OF THE ARCHAEAL INTRON ENCODED ENDONUCLEASE I-DmoI,. George H. Silva, Jacob Z. Dalgaard, Marlene Belfort, Patrick Van Roey, Wadsworth Center, New York State Dept. of Health, Albany, NY 12201-0509, USA

I-DmoI, is a 22-kDa endonuclease encoded by an intron in the 23S rRNA gene of the hyperthermophilic archaeon Desulfurococcus mobilis. The endonuclease is thermostable with peak activity in the 65-75^o C range in vitro and is capable of cleaving the intronless allele of the 23S rRNA gene. It recognizes an asymmetric target sequence and makes a staggered double-strand cut proximal to the intron insertion site generating 4-nucleotide 3'-OH extensions. A minimal binding site of 14-20 base pairs has been determined. I-DmoI, requires a Mg⁺⁺ co-factor for catalysis, but will readily bind DNA in its absence. I-DmoI, contains the common LAGLI-DADG motif found in eukaryotic intron endonucleases. The motif consists of two twelve amino-acid sequences separated by about 100 non-essential residues. This motif is seen in proteins associated with DNA cleavage, RNA maturation and protein processing. Crystals of I-DmoI, have been grown by the hanging drop method in PEG 3350, lithium sulfate and Tris.HCl pH 8.5. These crystals belong to the monoclinic space group C2 with unit cell dimensions a = 96.57 Å, b = 37.69 Å, c = 56.72 Å, ß = 114.09^o and V = 206,445 ų. Assuming two molecules per asymmetric unit yields a V_M ~ 2.35 ų/D, corresponding to about 48% solvent content. Native diffraction data have been measured to 2.3 Å resolution using flash cooling techniques. In addition, three possible derivative data sets have been obtained and are currently being used in phasing. Progress in the structure determination will be presented.