PRELIMINARY CHARACTERIZATION OF ECORI(ASTERISK)-DNA CO-CRYSTALS - INCOMPLETE FACTORIAL DESIGN OF OLIGONUCLEOTIDE SEQUENCES

A full understanding of the sequence specificity of EcoRI endonuclease requires structural information on complexes where the DNA contains o ne 'incorrect' base pair; historically, these sites are referred to as EcoRI sites. They are inherently asymmetric, unlike the canonical Ec oRI site, GAATTC, which possesses a twofold axis of rotational symmetr y. All previously determined DNA-EcoRI complexes incorporated this sym metry axis into the space group, requiring the design of 'new' oligonu cleotides to produce an asymmetric unit appropriate to an EcoRI compl ex. The incomplete factorial approach of Carter & Carter [Carter & Car ter (1979). J. Biol. Chem. 254, 12219-12223.] was used to design the D NA sequence. Factors included the location and type of EcoRI substitu tion and the length and AT richness of the sequences on both sides of the RI site. Cocrystals were obtained with several sequences, includin g one with TCGTGGACTTCGTG. Diffraction data were collected from one cr ystal of this complex to 3.2 Angstrom resolution; the unit-cell parame ters are a = b = 123.8 and c = 148.9 Angstrom and the space group is P 3(2)21. Unit-cell and space-group information was also obtained for th e EcoRI sites AAATTC, GGATTC and GAGTTC. These experiments demonstrat ed the need for a rapid, economical method that would distinguish DNA- protein cocrystals from crystals of protein only. This can be readily achieved with a single small crystal and a staining method based on me thylene blue and methyl violet, which stain DNA and protein, respectiv ely.