Biomedical subjects

W Guschlbauer

Publications and source records attributed to W Guschlbauer.

At least 19 recordsLinked to original sources

PubMed1997

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.

2-Aminopurine↗

PubMed1996

"Small is beautiful": major modifications in DNA structure or dynamics by small substituents or ligands.

This short review assembles the contributions of the author's laboratory to the structural aspects of DNA. DNA was modified by small ligands and/or substituents. There are three aspects to this work: a) Protonation of guanosine and DNA and the formation of triple- and quadruple-strands of guanosine, its nucleotides, their polymers and DNA. b) Substitution of the 2'-position of deoxyribose by the most polar atom, fluorine: studies on 2'-deoxy-2'-fluro-nucleosides, -nucleotides and their polymers, studied both by structural and biological methods. c) The effect of introducing the methyl group in the large groove of DNA: NMR studies of oligonucleotides containing N6-methylated adenine residues, and enzymatic and molecular biology work on Dam methylase are reported.

Adenine↗

PubMed1995

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.

2-Aminopurine↗

PubMed1995

Kinetic studies of MvaI DNA methyltransferase interaction with modified oligonucleotide duplexes.

We have measured steady-state kinetics of a N4-cytosine methylase, M.MvaI, using as substrates modified non-selfcomplementary tetradecanucleotide duplexes containing the CCWGG target sequence. The inner or outer localisation of the dI residue in the MvaI recognition site seems to be of little importance since the specificity constants kcat/KM are only 2 to 7 fold smaller than that of the canonical substrate. Replacement of dG residues by dI in both strands resulted in a 25 to 60-fold decrease of the specificity constant. Modifications of the phosphate backbone or opening of the sugar ring of one of the dG residues had only little influence on the action of M.MvaI. The enzyme appears to be rather tolerant to different kinds of modifications in its substrate in the mainor groove.

Base Sequence↗

PubMed1995

Use of UV spectroscopy for the study of nucleic acid cleavage by E. coli RNase H and restriction endonucleases.

A one-step spectrophotometric method for monitoring of nucleic acid cleavage by ribonuclease H from E. coli and type II restriction endonucleases has been proposed. It is based on recording of the increase in the UV absorbance at 260 nm during the course of enzymatic reaction. Duplexes stable under the reaction conditions were chosen as substrates for the enzymes being studied. In order to obtain duplex dissociation following their cleavage by the enzyme appreciate temperature conditions were selected. The spectrophotometric method may be applied for rapid testing of the nuclease activity in protein preparations as well as for precise quantitative analysis of nucleic acid degradation by enzymes. This method may be successfully employed in kinetic studies of nucleic acid-protein interactions.

Base Sequence↗

PubMed1994

Two subunits of EcoRII restriction endonuclease interact with two DNA recognition sites.

The cleavage of a 14 base pair DNA duplex containing one EcoRII recognition site by EcoRII restriction endonuclease (R.EcoRII) was studied in single turnover experiments with varying enzyme concentrations in the micromolar range. The reaction rate increased with enzyme concentration until a ratio of one dimeric R.EcoRII enzyme to two double stranded oligonucleotide molecules. Excess of R.EcoRII lead to inhibition of cleavage. Maximum cleavage was also found with pBR322 DNA containing six EcoRII recognition sites at a ratio of one dimeric enzyme to two EcoRII recognition sites of the plasmid DNA. At higher ratios inhibition was observed. These observations indicate that the active enzyme complex is formed when two subunits of the enzyme interact with two R.EcoRII recognition sites.

Base Composition↗

PubMed1993

Dam methyltransferase from Escherichia coli: sequence of a peptide segment involved in S-adenosyl-methionine binding.

DNA adenine methyltransferase (Dam methylase) has been crosslinked with its cofactor S-adenosyl methionine (AdoMet) by UV irradiation. About 3% of the enzyme was radioactively labelled after the crosslinking reaction performed either with (methyl-3H)-AdoMet or with (carboxy-14C)-AdoMet. Radiolabelled peptides were purified after trypsinolysis by high performance liquid chromatography in two steps. They could not be sequenced due to radiolysis. Therefore we performed the same experiment using non-radioactive AdoMet and were able to identify the peptide modified by the crosslinking reaction by comparison of the separation profiles obtained from two analytical control experiments performed with 3H-AdoMet and Dam methylase without crosslink, respectively. This approach was possible due to the high reproducibility of the chromatography profiles. In these three experiments only one radioactively labelled peptide was present in the tryptic digestions of the crosslinked enzyme. Its sequence was found to be XA-GGK, corresponding to amino acids 10-14 of Dam methylase. The non-identified amino acid in the first sequence cycle should be a tryptophan, which is presumably modified by the crosslinking reaction. The importance of this region near the N-terminus for the structure and function of the enzyme was also demonstrated by proteolysis and site-directed mutagenesis experiments.

Amino Acid Sequence↗

PubMed1993

The role of the preserved sequences of Dam methylase.

We have undertaken a site directed mutational analysis of two of the preserved regions in the amino acid sequence of Dam methylase in order to characterize their role. Mutations in region IV (sequence DPPY) abolish catalytic activity and greatly affect AdoMet crosslinking. Mutants in region III display a lowered specific activity with an unchanged AdoMet crosslinking capacity. We have also made a series of deletions both at the N and C terminal parts of the protein, which have been found to provide inactive enzyme. We discuss the significance of these results for the understanding of the functional properties of the enzyme.

Amino Acid Sequence↗

PubMed1992

Improved plasmids containing the Escherichia coli dam gene under the control of the tac promoter.

We report the construction of a series of plasmids containing the dam gene under the control of the tac promoter. Cells containing these plasmids produce about 8 to 10-fold more Dam methyltransferase (Mtase) than the previously used plasmid pTP166 and avoid the use of a high temperature step necessary for the expression of Dam Mtase in the plasmid pDOX1 and thus allow its use for the study of thermosensitive mutants.

Cloning, Molecular↗

PubMed1992

Expression of Escherichia coli dam gene in Bacillus subtilis provokes DNA damage response: N6-methyladenine is removed by two repair pathways.

The dam gene of Escherichia coli encodes a DNA methyltransferase that methylates the N6 position of adenine in the sequence GATC. It was stably expressed from a shuttle vector in a repair- and recombination-proficient strain of Bacillus subtilis. In this strain the majority of plasmid DNA molecules was modified at dam sites whereas most chromosomal DNA remained unmethylated during exponential growth. During stationary phase the amount of unmethylated DNA increased, suggesting that methylated bases were being removed. An ultraviolet damage repair-deficient mutant (uvrB) contained highly methylated chromosomal and plasmid DNA. High levels of Dam methylation were detrimental to growth and viability of this mutant strain and some features of the SOS response were also induced. A mutant defective in the synthesis of adaptive DNA alkyltransferases and induction of the adaptive response (ada) also showed high methylation and properties similar to that of the dam gene expressing uvrB strain. When protein extracts from B. subtilis expressing the Dam methyltransferase or treated with N-methyl-N'-nitro-N-nitroso-guanidine were incubated with [3H]-labelled Dam methylated DNA, the methyl label was bound to two proteins of 14 and 9 kD. Some free N6-methyladenine was also detected in the supernatant of the incubation mixture. We propose that N6-methyladenine residues are excised by proteins involved in both excision (uvrB) and the adaptive response (ada) DNA repair pathways in B. subtilis.

Adenine↗

PubMed1992

[Biological actions and therapeutic perspectives of double stranded polyribonucleotides: a reappraisal].

Double-stranded polynucleotides, which are composed of two complementary homopolyribonucleotides containing no genetic information, are synthetic molecules capable of mimicking the action of natural double-stranded RNA or viral RNA on cells. Double-stranded polyribonucleotides act as an alarm system alerting the cell to the presence of an external aggression, e.g. a viral attack. In addition, polyribonucleotides have a more active function in that they trigger cell defense processes through activation of a family of genes, of which some encode cytokines, activation of cytoplasmic enzymes involved in antiviral mechanisms or signal transduction, and activation of nonspecific immune responses. Double-stranded polyribonucleotides containing one mismatched base pair per helix have been found to be especially interesting. The best known example is poly(I).poly(C12U), also called ampligen. Poly(I).poly(C12U) is capable, in experimental models, of limiting the development of viruses (including HIV), reducing tumor growth, eliminating metastases, and, according to one report, preventing steady declines in T-cell counts in HIV-positive patients. Therapeutic doses used in the USA as an experimental drug induced little toxicity. In vitro, poly(I).poly(C12U) acts synergistically with interferon, interleukin 2 or AZT, suggesting that these latter drugs may be effective in lower, less toxic doses when used in combination with poly(I).poly(C12U). The therapeutic activity of poly(I).poly(C12U) holds promise. More extensive prospective studies of this agent are warranted.

Cytomegalovirus Infections↗

PubMed1991

The solution structure of a DNA hairpin containing a loop of three thymidines determined by nuclear magnetic resonance and molecular mechanics.

We have determined by two-dimensional nuclear magnetic resonance studies and molecular mechanics calculations the three-dimensional solution structure of a 21 residue oligonucleotide capable of forming a hairpin structure with a loop of three thymidine residues. This structure is in equilibrium with a duplex form. At 33 degrees C, low ionic strength and in the presence of MgCl2 the hairpin form dominates in solution. Six Watson-Crick base pairs are formed topped by the loop structure. The residues 1-3 and 18-21 are not complementary and form dangling ends. Distance constraints have been derived from nuclear Overhauser enhancement measurements. These, together with molecular mechanics calculations, have been used to determine the structure. We do not observe stacking of thymidine residues either over the 3' or the 5' end of the stem.

Base Sequence↗

PubMed1991

Allosteric and catalytic binding of S-adenosylmethionine to Escherichia coli DNA adenine methyltransferase monitored by 3H NMR.

Adenine methylation of GATC sequences in DNA is carried out by the DNA adenine methyltransferase with the methyl group source being the cofactor S-adenosylmethionine. We report 3H NMR studies on the interaction of DNA adenine methyltransferase with S-adenosylmethionine and the reaction when the ternary complex is formed with an oligonucleotide containing a GATC site. The methylation reaction was also studied in the presence of a competitive inhibitor and this showed two successive stages involved in the methylation and two sites of binding for S-adenosylmethionine.

Allosteric Site↗

PubMed1991

Solution structure of an oncogenic DNA duplex containing a G.A mismatch.

The DNA duplex 5'-d(GCCACAAGCTC).d(GAGCTGGTGGC), which contains a central G.A mismatch has been studied by one and two-dimensional NMR techniques. The duplex corresponds to the sequence 29-39 of the K-ras gene. The mismatch position is that of the first base of the Gly12 codon, a hot spot for mutations. The observed NOEs of the nonexchangeable protons show that both of the bases of the mismatched pair are intrahelical over a wide range of pH. However, the structure of the G.A mispair and the conformation of the central part of the duplex change with pH. This structural change shows a pK of 6.0. At low pH, the G.A bases are base paired with hydrogen bonds between the keto group of the G residue and the amino group of the A residue and, secondly, between the N7 of the G and a proton on N1 of A. This causes the G residue to adopt a syn conformation. On raising the pH, the N1-H proton of the protonated A residue is removed, and the base pair rearranges. In the neutral G.A base pair both residues adopt an anti conformation, and the mismatch is stabilized by hydrogen bonds. Our results on the exchangeable and A(H2) protons of the mismatched pair indicate a shift from a classical face-to-face two hydrogen-bonded structure to a slipped structure stabilized by bifurcated hydrogen bonds. This may be a particular characteristics of this oncogenic sequence in which the G.A error is poorly repaired.

Base Composition↗

PubMed1991

Structure and in vitro replication of DNA templates containing 7,8-dihydro-8-oxoadenine.

Adenine residues in DNA are oxidized under the action of ionizing radiation at the C-8 position to give 7,8-dihydro-8-oxoadenine. The formation of this lesion can be considered a cause of mutations and carcinogenesis. Oligodeoxyribonucleotides 39 and 47 bases long containing a single 7,8-dihydro-8-oxoadenine (8-hydroxyadenine) residue were synthesized by using nucleoside phosphoramidites. They were used as templates to study the copies obtained in vitro by the Klenow fragment and the thermostable Taq DNA polymerase. 7,8-Dihydro-8-oxoadenine does not block the replication and thymine is incorporated opposite the damage. The modifications of the DNA duplex conformation provoked by 7,8-dihydro-8-oxoadenine are minor. 1H-NMR spectroscopy shows that the duplex is in a B form, the sugar in a normal position in the helix and the modified base in the anti position. NMR confirms that 7,8-dihydro-8-oxoadenine exists predominantly in the keto form.

Adenine↗

PubMed1991

Crosslinking of Dam methyltransferase with S-adenosyl-methionine.

Highly purified DNA-adenine methyltransferase was irradiated in the presence of different concentrations of radiolabelled S-adenosyl-methionine (AdoMet) with a conventional Mineralight UV-lamp from several minutes up to 1 h while incubating in ice. Incorporation of radioactivity was monitored by electrophoresis of the crosslink between S-adenosyl-methionine and Dam methylase on SDS-polyacrylamide gels followed by fluorography. Crosslinking reached a maximum in presence of 10 microM S-adenosyl-methionine; it was inhibited in the presence of substances which competitively inhibit methylation of DNA by Dam methylase, like sinefungin or S-adenosyl-homocysteine, but not in the presence of non-inhibitors like ATP or S-isobutyl-adenosine. The crosslink obtained was resistant against a wide range of even drastic conditions commonly used in protein and peptide chemistry. Proteins, which do not bind S-adenosyl-methionine, as well as heat inactivated Dam methylase were not photolabelled. After limited proteolysis the radioactive label appeared only in certain of the peptides obtained. From Western blots carried out with polyclonal antibodies produced against a synthetic peptide corresponding in its sequence to amino acids 92-106 of the Dam methylase, the crosslinking of AdoMet could be tentatively mapped at a position after amino acid 106.

Binding, Competitive↗

PubMed1990

The double role of methyl donor and allosteric effector of S-adenosyl-methionine for Dam methylase of E. coli.

The turnover of DNA-adenine-methylase of E. coli strongly decreases when the temperature is lowered. This has allowed us to study the binding of Dam methylase on 14 bp DNA fragments at 0 degrees C by gel retardation in the presence of Ado-Met, but without methylation taking place. The enzyme can bind non-specific DNA with low affinity. Binding to the specific sequence occurs in the absence of S-adenosyl-methionine (Ado-Met), but is activated by the presence of the methyl donor. The two competitive inhibitors of Ado-Met, sinefungin and S-adenosyl-homocysteine, can neither activate this binding to DNA by themselves, nor inhibit this activation by Ado-Met. This suggests that Ado-Met could bind to Dam methylase in two different environments. In one of them, it could play the role of an allosteric effector which would reinforce the affinity of the enzyme for the GATC site. The analogues can not compete for such binding. In the other environment Ado-Met would be in the catalytic site and could be exchanged by its analogues. We have also visualized conformational changes in Dam methylase induced by the simultaneous binding of Ado-Met and the specific target sequence of the enzyme, by an anomaly of migration and partial resistance to proteolytic treatment of the ternary complex Ado-Met/Dam methylase/GATC.

Adenosine↗

PubMed1990

The abasic site as a challenge to DNA polymerase. A nuclear magnetic resonance study of G, C and T opposite a model abasic site.

An abasic site in DNA creates a strong block to DNA polymerase and is a mutagenic base lesion. In this study, we present structural and dynamic properties of duplex oligodeoxynucleotides containing G, C and T opposite a model abasic site studied by one and two-dimensional nuclear magnetic resonance spectroscopy. We have demonstrated that A opposite the abasic site was positioned within the helix as if paired with T, and that the A residue melted co-operatively with the surrounding helix. We report here that G opposite the abasic site is also observed to be predominantly intrahelical in a normal anti conformation at low temperature. With increasing temperature, the mobility of the G residue increases rapidly and apparently is in a "melted state" well before denaturation of the helix. At low temperature, two species are found for T opposite the abasic site; one, intrahelical, one extrahelical. These species are in slow exchange with one another on a proton nuclear magnetic resonance time-scale. The two species then move into fast exchange with increasing temperature and the proportion of the extra-helical form increases. When C is positioned opposite the abasic site, both the C residue and the abasic sugar are extrahelical, the helix collapses, and the adjacent G.C base-pairs stack over one another. On the basis of these observations, we propose a model that explains why the abasic site acts to block DNA replication. Further, we suggest an explanation for the observed polymerase preference for base selection at abasic sites.

Base Sequence↗