The fragments of genomics DNA of the nuclear polyhedrosis virus (NPV) containing genes of late viral proteins p10, p35, p39, were cloned, the promoter regions of this genes were used to design baculovirus transfer vectors. A double-promoter and triple-promoter baculovirus transfer vectors were obtained. Recombinant baculovirus vectors containing mammalian expression cassette with cytomegalovirus (CMV) promoter, the gene for green or red fluorescent protein, SV40pA and polylinker MCS were constructed for the delivery of foreign genes into mammalian cells.

High sequence selectivity in DNA-protein interactions was analyzed by measuring discrimination by Eco RI endonuclease between the recognition site GAATTC and systematically altered DNA sites. Base analogue substitutions that preserve the sequence-dependent conformational motif of the GAATTC site permit deletion of single sites of protein-base contact at a cost of +1 to +2 kcal/mol. However, the introduction of any one incorrect natural base pair costs +6 to +13 kcal/mol in transition state interaction energy, the resultant of the following interdependent factors: deletion of one or two hydrogen bonds between the protein and a purine base; unfavourable steric apposition between a group on the protein and an incorrectly placed functional group on a base; disruption of a pyrimidine contact with the protein; loss of some crucial interactions between protein and DNA phosphates; and an increased energetic cost of attaining the required DNA conformation in the transition state complex. Eco RI endonuclease thus achieves stringent discrimination by both "direct readout"(protein-base contracts) and "indirect readout"(protein-phosphate contacts and DNA conformation) of the DNA sequence.

The MunI restriction enzyme recognizes the palindromic hexanucleotide sequence C/AATTG (the '/' indicates the cleavage site). The crystal structure of its active site mutant D83A bound to cognate DNA has been determined at 1.7 A resolution. Base-specific contacts between MunI and DNA occur exclusively in the major groove. While DNA-binding sites of most other restriction enzymes are comprised of discontinuous sequence segments, MunI combines all residues involved in the base-specific contacts within one short stretch (residues R115-R121) located at the N-terminal region of the 3(10)4 helix. The outer CG base pair of the recognition sequence is recognized solely by R115 through hydrogen bonds made by backbone and side chain atoms to both bases. The mechanism of recognition of the central AATT nucleotides by MunI is similar to that of EcoRI, which recognizes the G/AATTC sequence. The local conformation of AATT deviates from the typical B-DNA form and is remarkably similar to EcoRI-DNA. It appears to be essential for specific hydrogen bonding and recognition by MunI and EcoRI.

The crystal structure of restriction endonuclease Bam HI complexed to DNA has been determined at 2.2 angstrom resolution. The DNA binds in the cleft and retains a B-DNA type of conformation. The enzyme, however, undergoes a series of conformational changes, including rotation of subunits and folding of disordered regions. The most striking conformational change is the unraveling of carboxyl-terminal alpha helices to form partially disordered "arms." The arm from one subunit fits into the minor groove while the arm from the symmetry related subunit follows the DNA sugar-phosphate backbone. Recognition of DNA base pairs occurs primarily in the major groove, with a few interactions occurring in the minor groove. Tightly bound water molecules play an equally important role as side chain and main chain atoms in the recognition of base pairs. The complex also provides new insights into the mechanism by which the enzyme catalyzes the hydrolysis of DNA phosphodiester groups.

To date all attempts to derive a phyletic relationship among restriction endonucleases (ENases) from multiple sequence alignments have been limited by extreme divergence of these enzymes. Based on the approach of Johnson et al.(1990), I report for the first time the evolutionary tree of the ENase-like protein superfamily inferred from quantitative comparison of atomic coordinates of structurally characterized enzymes. The results presented are in harmony with previous comparisons obtained by crystallographic analyses. It is shown that lambda-exonuclease initially diverged from the common ancestor and then two "endonucleolytic" families branched out, separating "blunt end cutters" from "5' four-base overhand cutters." These data may contribute to a better understanding of ENases and encourage the use of structure-based methods for inference of phylogenetic relationship among extremely divergent proteins. In addition, the comparison of three-dimensional structures of ENase-like domains provides a platform for further clustering analyses of sequence similarities among different branches of this large protein family, rational choice of homology modeling templates, and targets for protein engineering.

DNA methyltransferases modify specific cytosines and adenines within 2-6 bp recognition sequences. We used scanning force microscopy and gel shift analysis to show that M.HhaI, a cytosine C-5 DNA methyltransferase, causes only a 2 degree bend upon binding its recognition site. Our results are consistent with prior crystallographic analysis showing that the enzyme stabilizes an extrahelical base while leaving the DNA duplex otherwise unperturbed. In contrast, similar analysis of M.EcoRI, an adenine N6 DNA methyltransferase, shows an average bend angle of approximately 52 degrees. This distortion of DNA conformation by M.EcoRI is shown to be important for sequence-specific binding.

In baker's yeast (Saccharomyces cerevisiae), accumulation of the non-reducing disaccharide, trehalose, is triggered by stimuli that activate the heat-shock response. Previously, trehalose levels have been shown to be closely correlated with thermotolerance, suggesting a protective function of this substance. Genetic evidence in support of this view is presented in an accompanying paper [De Virgilio, C., Hottiger, T., Dominguez, J., Boller, T.& Wiemken, A.(1993) Eur. J. Biochem. 219, 179-186]. In this study, we have examined the effect of trehalose on the thermal stability of proteins, a parameter thought to be a major determinant of thermotolerance. Physiological concentrations of trehalose (up to 0.5 M) were found to efficiently protect enzymes of yeast (glucose-6P-dehydrogenase, phosphoglucose-isomerase) as well as enzymes of non-yeast origin (bovine glutamic dehydrogenase, EcoRI) against heat inactivation in vitro. Trehalose also reduced the heat-induced formation of protein aggregates. The disaccharide proved to be a compatible solute, as even at very high concentrations (up to 1 M) it did not significantly interfere with the activity of test enzymes. Trehalose was at least as good or better a protein stabilizer than any of a number of other compatible solutes (including sugars, polyalcohols and amino acids), while the structurally related trehalose-6P was devoid of any protective effect. Thermoprotection of enzymes by trehalose was evident even in solutions containing high concentrations of yeast protein or substrate. The data indicate that trehalose accumulation may increase the thermotolerance of yeast by enhancing protein stability in intact cells.

Type II restriction endonucleases are characterized by the remarkable specificity with which they cleave specific DNA sequences. Surprisingly, their protein sequences are in most cases unrelated, and no recurring structural motif has yet been identified. We have determined the structure of restriction endonuclease BamHI at 1.95 A resolution. BamHI shows striking resemblance to the structure of endonuclease EcoRI (refs 3, 4), despite the lack of sequence similarity between them. We also observe some curious differences between the two structures, and propose an evolutionary scheme that may explain them. The active site of BamHI is structurally similar to the active sites of EcoRI and EcoRV (ref. 5), but the mechanism by which BamHI activates a water molecule for nucleophilic attack may be different.

We have probed the contacts between EcoRI endonuclease and the central phosphate of its recognition site GAApTTC, using synthetic oligonucleotides containing single stereospecific Rp- or Sp-phosphorothioates (Ps). These substitutions produce subtle stereospecific effects on EcoRI endonuclease binding and cleavage. An Sp-Ps substitution in one strand of the DNA duplex improves binding free energy by -1.5 kcal/mol, whereas the Rp-Ps substitution has an unfavorable effect (+0.3 kcal/mol) on binding free energy. These effects derive principally from changes in the first order rate constants for dissociation of the enzyme-DNA complexes. The first order rate constants for strand scission are also affected, in that a strand containing Sp-Ps substitution is cleaved 2 to 3 times more rapidly than a strand containing a normal prochiral phosphate, whereas a strand containing Rp-Ps substitution is cleaved about 3 times slower than normal. As a result, single-strand substitutions produce pronounced asymmetry in the rates of cleavage of the two DNA strands, and this effect is exaggerated in an Rp,Sp-heteroduplex. Ethylation-interference footprinting indicates that none of the Ps substitutions cause any major change in contacts between endonuclease and DNA phosphates. When an Sp-Ps localizes P = O in the DNA major groove, a hydrogen-bonding interaction with the backbone amide-NH of Gly116 of the endonuclease is improved relative to that with a prochiral phosphate having intermediate P-O bond order and delocalized charge.

A 0.7-ns molecular dynamics simulation of the DNA-EcoRI complex in a 7.0-A solvent shell indicated a stable behavior of the system. No significant evaporation or smearing of the solvent's outer boundary occurred. The structure and the intermolecular interactions were found to be well maintained during the simulation. The interaction pattern in the simulation was found to be very similar to that in the crystal structure. Most of the specific interactions between the DNA and the protein were found to be enhanced in the simulation compared to that in the crystal structure as a result of improved interaction geometry. The nonspecific interactions were found to be stronger than the specific ones. The specific interactions between the N7 atoms of Gua(4) or Ade(5) or Ade(6) and the protein were found to be present over almost the entire time of the simulation, whereas hydrogen bonds involving the amino groups of the Ade(5) and Ade(6) with the protein were found to be relatively weaker, with lower probability and shorter lifetime. The time evolution of the root mean square deviations of the DNA and the protein were highly correlated even at the later part of the simulation, showing the tight binding between them. Several long-lived water bridges were found between the DNA backbone atoms and the protein and also between the two protein monomers, which increased the overall stability of the complex. The two protein monomers were found to interact strongly with each other. The energy of the DNA kink deformation was estimated as approximately 31 kcal/mol.

The structure of I-Crel provides the first view of a protein encoded by a gene within an intron. This endonuclease recognizes a long DNA site approximately 20 base pairs in length and facilitates the lateral transfer of that intron. The protein exhibits a DNA-binding surface consisting of four antiparallel beta-strands that form a 20 A wide groove which is over 70 A long. The architecture of this fold is different from that of the TATA binding protein, TBP, which also contains an antiparallel beta-saddle. The conserved LAGLIDADG motif, which is found in many mobile intron endonucleases, maturases and inteins, forms a novel helical interface and contributes essential residues to the active site.