Dam methyltransferase from Escherichia coli: kinetic studies using modified DNA oligomers: nonmethylated substrates.

Steady-state kinetics of the N6-adenine Dam methyltransferase have been measured using as substrates non-self-complementary tetradecanucleotide duplexes that contain the GATC target sequence. Modifications in the GATC target sequence of one or both of the strands included substitution of guanine by hypoxanthine, thymine by uracil or 5-ethyl-uracil and adenine by diamino-purine (2-amino-adenine). Thermodynamic parameters for the 14-mer duplexes were also determined. DNA methylation of duplexes containing single dl for dG substitution of the Dam recognition site was little perturbed compared with the canonical substrate. Replacement of dG residues by dl in both strands resulted in a decrease of the specificity constant. Substitution in both strands appears to be cumulative. Substitution of the methyl-accepting adenine residues by 2-amino-adenine resulted in surprisingly little perturbation. Dam methyltransferase is rather tolerant to different substitutions. The results show much less spread than those for the analogous hemimethylated substrates studied previously (Marzabal et al., 1995). The absence of the methylation marker appears to be deleterious to the specificity of the transition state of the active complex, while the binding of the DNA substrate to the enzyme appears to be mostly determined by the thermodynamic stability of the DNA duplex.

Dam methylase from Escherichia coli: kinetic studies using modified DNA oligomers: hemimethylated substrates.

We have measured steady-state kinetics of the N6-adenine methyltransferase Dam Mtase using as substrates non-selfcomplementary tetradecamer duplexs (d[GCCGGATCTAGACG]-d[CGTCTAGATCC-GGC]) containing the hemimethylated GATC target sequence in one or the other strand and modifications in the GATC target sequence of the complementary strands. Modifications included substitution of guanine by hypoxanthine (I), thymine by uracil (U) or 5-ethyl-uracil (E) and adenine by 2,6-diamino-purine (D). Thermodynamic parameters were obtained from the concentration dependence of the melting temperature (Tm) of the duplexes. Large differences in DNA methylation of duplexes containing single dI for dG substitution of the Dam recognition site were observed compared with the canonical substrate, if the substitution involved the top strand (on the G.C rich side). Substitution in either strand by uracil (dU) or 5-ethyluracil (dE) resulted in small perturbation of the methylation patterns. When 2,6-diamino-purine (dD) replaced the adenine to be methylated, small, but significant methylation was observed. The kinetic parameters of the methylation reaction were compared with the thermodynamic free energies and significant correlation was observed.

A site-directed mutagenesis study to identify amino acid residues involved in the catalytic function of the restriction endonuclease EcoRV.

We have used site-directed mutagenesis of the EcoRV restriction endonuclease to change amino acid side chains that have been shown crystallographically to be in close proximity to the scissile phosphodiester bond of the DNA substrate. DNA cleavage assays of the resulting mutant proteins indicate that the largest effects on nucleolytic activity result from substitution of Asp74, Asp90, and Lys92. We suggest on the basis of structural information, mutagenesis data, and analogies with other nucleases that Asp74 and Asp90 might be involved in Mg2+ binding and/or catalysis and that Lys92 probably stabilizes the pentacovalent phosphorus in the transition state. These amino acids are part of a sequence motif, Pro-Asp...Asp/Glu-X-Lys, which is also present in EcoRI. In both enzymes, it is located in a structurally similar context near the scissile phosphodiester bond. A preliminary mutational analysis with EcoRI indicates that this sequence motif is of similar functional importance for EcoRI and EcoRV. On the basis of these results, a proposal is made for the mechanism of DNA cleavage by EcoRV and EcoRI.

Mg2+ confers DNA binding specificity to the EcoRV restriction endonuclease.

The EcoRV mutant D90A which carries an amino acid substitution in its active center does not cleave DNA. Therefore, it is possible to perform DNA binding experiments with the EcoRV-D90A mutant both in the absence and in the presence of Mg2+. Like wild-type EcoRV [Taylor et al. (1991) Biochemistry 30, 8743-8753], it does not show a pronounced specificity for binding to its recognition site in the absence of Mg2+ as judged by the appearance of multiple shifted bands in an electrophoretic mobility shift assay with a 377-bp DNA fragment carrying a single EcoRV recognition sequence. In the presence of Mg2+, however, only one band corresponding to a 1:1 complex appears even with a high excess of protein over DNA. This complex most likely is the specific one, because its formation is suppressed much more effectively by a 13-bp oligodeoxynucleotide with an EcoRV site than by a corresponding oligodeoxynucleotide without an EcoRV site. The preferential interaction of the EcoRV-D90A mutant with specific DNA in the presence of Mg2+ was also demonstrated directly: a 20-bp oligodeoxynucleotide with an EcoRV site is bound with KAss = 4 x 10(8) M-1, while a corresponding oligodeoxynucleotide without an EcoRV site is bound with KAss less than or equal to 1 x 10(5) M-1. From these data it appears that Mg2+ confers DNA binding specificity to this mutant by lowering the affinity to nonspecific sites and raising the affinity to specific sites as compared to binding in the absence of Mg2+. It is concluded that this is also true for wild-type EcoRV.

Assessing staff morale through a staff opinion survey.

This paper reports the results of a staff opinion survey conducted at a 309-bed Victorian public hospital. The reasons for the survey were to measure staff morale and attitudes across all the departments of the hospital. Staff members were able to participate in the survey, by completing a questionnaire which was distributed to all staff. The overall results indicate that staff members of Frankston Hospital consider they operate in a healthy working environment. Staff from some departments within the hospital responded to several questions in a manner that required further investigation. These have since been actively discussed and decisions taken to overcome them.

Site-directed mutagenesis studies with EcoRV restriction endonuclease to identify regions involved in recognition and catalysis.

Guided by the X-ray structure analysis of a crystalline EcoRV-d(GGGATATCCC) complex (Winkler, in preparation), we have begun to identify functionally important amino acid residues of EcoRV. We show here that Asn70, Asp74, Ser183, Asn185, Thr186, and Asn188 are most likely involved in the binding and/or cleavage of the DNA, because their conservative substitution leads to mutants of no or strongly reduced activity. In addition, C-terminal amino acid residues of EcoRV seem to be important for its activity, since their deletion inactivates the enzyme. Following the identification of three functionally important regions, we have inspected the sequences of other restriction and modification enzymes for homologous regions. It was found that two restriction enzymes that recognize similar sequences as EcoRV (DpnII and HincII), as well as two modification enzymes (M.DpnII and, in a less apparent form, M.EcoRV), have the sequence motif -SerGlyXXXAsnIleXSer- in common, which in EcoRV contains the essential Ser183 and Asn188 residues. Furthermore, the C-terminal region, shown to be essential for EcoRV, is highly homologous to a similar region in the restriction endonuclease SmaI. On the basis of these findings we propose that these restriction enzymes and to a certain extent also some of their corresponding modification enzymes interact with DNA in a similar manner.

Accuracy of the EcoRI restriction endonuclease: binding and cleavage studies with oligodeoxynucleotide substrates containing degenerate recognition sequences.

We have synthesized a series of 18 nonpalindromic oligodeoxynucleotides that carry all possible base changes within the recognition sequence of EcoRI. These single strands can be combined with their complementary single strands to obtain all possible EcoRI sequences (left), or they can be combined with a single strand containing the canonical sequence to obtain double strands with all possible mismatches within the recognition sequence (right): (sequence; see text) The rate of phosphodiester bond cleavage of these oligodeoxynucleotides by EcoRI was determined in single-turnover experiments under normal buffer conditions in order to find out to what extent the canonical recognition site can be distorted and yet serve as a substrate for EcoRI. Our results show that oligodeoxynucleotides containing mismatch base pairs are in general more readily attacked by EcoRI than oligodeoxynucleotides containing EcoRI sites and that the rates of cleavage of the two complementary strands of degenerate oligodeoxynucleotides are quite different. We have also determined the affinities of these oligodeoxynucleotides to EcoRI. They are higher for oligodeoxynucleotides carrying a mismatch within the EcoRI recognition site than for oligodeoxynucleotides containing an EcoRI site but otherwise do not correlate with the rate with which these oligodeoxynucleotides are cleaved by EcoRI. Our results allow details to be given for the probability of EcoRI making mistakes in cleaving DNA not only in its recognition sequence but also in sequences closely related to it. Due to the fact that the rates of cleavage in the two strands of a degenerate sequence generally are widely different, these mistakes are most likely not occurring in vivo, since nicked intermediates can be repaired by DNA ligase.