Clustering of null mutations in the EcoRI endonuclease.

EcoRI endonuclease mutants were isolated in a methylase-deficient background following in vitro hydroxylamine mutagenesis of plasmid pKG2 (Kuhn et al.: Gene 44:253-263, 1986). Mutants which survived high-level endonuclease expression (IPTG induction) were termed null mutants. Sixty-two of 121 null mutants tested by Western blot contained normal levels of endonuclease cross-reacting protein. The complete endonuclease gene was sequenced for 27 null mutants. This group was found to consist of 20 single base-change missense mutations, 6 double mutations, and 1 triple mutation. Ten of the 20 single mutations were clustered between residues 139 and 144. When examined with respect to the structure of the EcoRI-DNA complex (McClarin et al.: Science 234:1526-1541, 1986), these alterations were found to fall predominantly into two classes: substitutions at the protein-DNA interface or substitutions at the protein-protein (dimer) interface. Protein from several of the mutants was purified and sized by using HPLC. Wild-type EcoRI endonuclease and protein from three of the DNA interface mutations (Ala139----Thr, Gly140----Ser, Arg203----Gln) appeared to be dimeric, while protein from subunit interface mutations (Glu144----Lys, Glu152----Lys, Gly210----Arg) migrated as monomers.

Structure of the DNA-Eco RI endonuclease recognition complex at 3 A resolution.

The crystal structure of the complex between Eco RI endonuclease and the cognate oligonucleotide TCGCGAATTCGCG provides a detailed example of the structural basis of sequence-specific DNA-protein interactions. The structure was determined, to 3 A resolution, by the ISIR (iterative single isomorphous replacement) method with a platinum isomorphous derivative. The complex has twofold symmetry. Each subunit of the endonuclease is organized into an alpha/beta domain consisting a five-stranded beta sheet, alpha helices, and an extension, called the "arm," which wraps around the DNA. The large beta sheet consists of antiparallel and parallel motifs that form the foundations for the loops and alpha helices responsible for DNA strand scission and sequence-specific recognition, respectively. The DNA cleavage site is located in a cleft that binds the DNA backbone in the vicinity of the scissile bond. Sequence specificity is mediated by 12 hydrogen bonds originating from alpha helical recognition modules. Arg200 forms two hydrogen bonds with guanine while Glu144 and Arg145 form four hydrogen bonds to adjacent adenine residues. These interactions discriminate the Eco RI hexanucleotide GAATTC from all other hexanucleotides because any base substitution would require rupture of at least one of these hydrogen bonds.

Molecular basis for the transfer of nicotinamide adenine dinucleotide among dehydrogenases.

NADH is transferred directly from one dehydrogenase enzyme site to another without intervention of the aqueous solvent whenever the two dehydrogenases are of opposite chiral specificity as regards the C4 H of NADH which is transferred in the catalyzed reduction reaction. When both enzymes catalyze the transfer of hydrogen from the same face of the nicotinamide ring, direct enzyme-enzyme transfer of NADH is not possible [Srivastava, D. K., & Bernhard, S. A. (1984) Biochemistry 23, 4538-4545; Srivastava, D. K., & Bernhard, S. A. (1985) Biochemistry (preceding paper in this issue)]. Utilizing an advanced computer graphics facility, and the known three-dimensional coordinates for three dehydrogenases, we have investigated the feasibility of various aspects of the direct transfer of dinucleotide from the site of one enzyme to the site of the other. The facile passage of the coenzyme through the first enzyme site requires an open protein conformation, characteristic of the apoenzyme rather than the holoenzyme structure. Since two dehydrogenases of the same chirality bind coenzyme in the same conformation, the direct transfer of coenzyme from one site to the other is impossible due to the restriction in molecular rotation of the coenzyme in the path of transfer from one binding site to the other; therefore, coenzyme can only be transferred from one dehydrogenase site to another site via the intermediate dissociation of coenzyme into the aqueous milieu. In contrast, when an A dehydrogenase and a B dehydrogenase are juxtaposed, it is stereochemically feasible to transfer the nicotinamide ring from its specific binding site in one enzyme to the site in the other.(ABSTRACT TRUNCATED AT 250 WORDS)