Biomedical subjects

W Guschlbauer

Publications and source records attributed to W Guschlbauer.

At least 37 records · Page 2Linked to original sources

PubMed1990

Four-stranded nucleic acid structures 25 years later: from guanosine gels to telomer DNA.

The subject of four-stranded nucleic acid structures is reviewed. Studies on gels formed by guanosine and its analogues have provided appropriate models for the structures of poly(I) and poly(G). The stabilizing influence of certain cations, in particular K+, on Guo-5'-P gels and poly(I) is discussed in the light of recent data on selective K+ stabilization of telomeric DNA structures. The topological possibilities these dG containing sequences could adopt are discussed. In particular the role of the glycosidic linkage (anti/syn), the polarity of the strands and the orientation of the G-tetrad stacks is highlighted.

Base Sequence↗

PubMed1989

The GTm6AC sequence is overwound and bent.

By a combination of distance constraints obtained from NMR spectra and molecular mechanics calculations we have determined the three dimensional structure of the self-complementary decanucleotide d(CGCGTm6ACGCG). Methylation of an adenine at a position 3' to T induces significant conformational changes relative to B-DNA. This arises from the close proximity of the four methyl groups in the large groove in the centre of the sequence. The helical twist between the two T.m6A base pairs is found to be 45 degrees, as for D-DNA, and is accompanied by a high negative value of the wedge roll angle between these base pairs. The overall nonzero wedge roll observed shows that the helix is bent. These constraints appear to be material for the absence of the sequence T-m6A in natural DNAs.

Adenine↗

PubMed1989

Abasic frameshift in DNA. Solution conformation determined by proton NMR and molecular mechanics calculations.

We have determined the three-dimensional structure of a non-self-complementary oligodeoxynucleotide duplex that contains a model abasic site. The duplex contains six GC base pairs plus the abasic site at the center of one strand and corresponds to an abasic frameshift. Two-dimensional NMR studies on the nonexchangeable protons show that the guanine bases on either side of the abasic site are stacked over each other and that the abasic site is rotated out of the helix. Close proton-proton interactions are observed between the H4' proton of the abasic site and sugar protons of the guanosine in the 5' direction, which allows the position of the free sugar to be well-defined. NOE buildup curves from NOESY spectra recorded at very short mixing times were used to calculate a set of interproton distances. This data set was incorporated into the refinement of the oligonucleotide structure by molecular mechanics calculations. Two conformations that differ in the sugar conformation of the guanosine next to the abasic site in the 3' direction were necessary to fit all the NMR data. One of these two conformations could only be stabilized by addition of counterions at specific sites.

Chemical Phenomena↗

PubMed1989

Preferential site-specific hemimethylation of GATC sites in pBR322 DNA by Dam methyltransferase from Escherichia coli.

The methylation pattern of the 22 GATC sites of pBR322 (dam-) by Dam methyltransferase from Escherichia coli has been studied. Preferential hemimethylation took place at positions 3042 and 349. It was found that these preferential methylations were the same in supercoiled circular and linear DNAs. The flanking regions of these preferentially methylated sites contain three G.C pairs on one side and two A.T pairs and one G.C pair on the other. This preferential methylation was confirmed on a 126-base pair oligonucleotide containing two GATC sites with different flanking sequences. The next sites methylated were, in both cases, the first GATC site on the A.T-rich side, although the orientation was different. The rapid methylation of a second and third neighboring GATC site on the same plasmid suggests a processive mechanism. The implications of the orientation of hemimethylation are discussed in the context of the recognition of a palindromic target site by a monomeric DNA-binding protein.

Chromosome Mapping↗

PubMed1988

The DNA and S-adenosylmethionine-binding regions of EcoDam and related methyltransferases.

Previous comparison of the amino acid sequences of the GATC-methylating Escherichia coli Dam methyltransferase (MTase) with those of other adenine MTases (M.EcoRV, M.DpnII and T4Dam) localized four conserved regions. Regions III and IV have similarities with many other MTases. The sequence DPPY (or NPPY) is always present in region IV. It was suggested to be the AdoMet binding site. Publication of the nucleotide and amino acid sequences of M.CviBIII, M.DpnA and MutH give further credence to this assignment: M.DpnA, which also methylates GATC, has strong similarities with regions III and IV; M.CviBIII, a cytosine methylase, has a characteristic NPPY sequence in region IV, and only limited resemblance in region III; MutH, the GATC-specific endonuclease in DNA mismatch repair, has significant similarities uniquely in region III. The presently available evidence suggests that region III is the GAT(C) binding site and region IV is the AdoMet binding site. This hypothesis is strengthened by recent genetic findings.

Amino Acid Sequence↗

PubMed1987

An abasic site in DNA. Solution conformation determined by proton NMR and molecular mechanics calculations.

We have determined the three-dimensional structure of a non-selfcomplementary nonanucleotide duplex which contains an abasic (apyrimidinic) site in the centre, i.e. a deoxyribose residue opposite an adenosine. The majority of the base and sugar proton resonances were assigned by NOESY, COSY and 2DQF spectra in D2O and H2O. We have measured the initial slope of buildup of NOEs in NOESY spectra at very short mixing times (25 to 50 ms), and from these were able to establish interproton distances for the central part of the duplex. We propose a different strategy for proton-proton distance determinations which takes into account the observed variations in correlation times for particular proton-proton vectors. A set of 31 measured interproton distances was incorporated into the refinement of the oligonucleotide structure by molecular mechanics calculations. Two structures were obtained which retain all aspects of a classical B DNA in which the unpaired adenine and the abasic deoxyribose lie inside the helix. We observe that the non-hydrogen bonded adenine is held well in the helix, the Tm of this base being the same as that of the A.T base pairs in the same duplex.

Algorithms↗

PubMed1987

A two-dimensional 1H-NMR study of the dam methylase site: comparison between the hemimethylated GATC sequence, its unmethylated analogue and a hemimethylated CATG sequence. The sequence dependence of methylation upon base-pair lifetimes.

We report two-dimensional NOE (NOESY) spectra on the sequence d(GCGATCATGG).d(CCATGATCGC) which contains the unmethylated dam site. As expected the DNA adopts a B-form conformation but appears to be distorted at the TG step of the second strand. This distorsion, probably bending, is not seen on the opposite strand. When the first strand is methylated on adenine in the GATC or CATG sequence the NOESY spectra indicate little or no change in the conformation. However the single strand-duplex exchange is slowed down to the slow-exchange region on a proton NMR time scale. We have assigned the exchangeable imino and cytidine amino resonances of the three duplexes. From the imino linewidths as a function of temperature, we observe that the unmethylated and the hemimethylated Gm6ATC duplexes melt normally from the ends. However, this is not so for the hemimethylated Cm6ATG duplex which, apart from the terminal base pairs, melts cooperatively and at higher temperature. In spectra recorded in H2O a second duplex is observed, for the Gm6ATC sequence, which we have not been able to identify. It is however unlikely to be a hairpin structure. Ultraviolet-melting curves also indicate the presence of two transitions for this duplex. The effect of methylation upon base-pair lifetimes has been studied by comparing the above three duplexes. Little effect is observed upon methylation in the GATC sequence but a drastic increase in the lifetimes of all base pairs is observed upon methylation in the CATG sequence.

Base Composition↗

PubMed1987

The GATATC-modification enzyme EcoRV is closely related to the GATC-recognizing methyltransferases DpnII and dam from E. coli and phage T4.

The amino acid sequence of EcoRV DNA methyltransferase which methylates the amino group of the 5'-adenine residue of the target sequence GATATC has been found to be closely related to that of three other adenine methyltransferases, DpnII, dam and damT4, the target sequence of which is GATC. Despite large differences on the DNA level, the four sequences show four blocks of homologies. One of these blocks has the sequence DVYXDPPY and is found with little modification in numerous other DNA methyltransferases. It is speculated that it could be the binding site of the methyl donor, S-adenosylmethionine. On the other hand, the identification of a DNA-binding region is more tenuous. As expected, no analogies with (dimeric) repressors and cro proteins which have the characteristic helix-turn-helix motif have been observed.

Amino Acid Sequence↗

PubMed1987

Comparison of the conformation of an oligonucleotide containing a central G-T base pair with the non-mismatch sequence by proton NMR.

We have recorded NOESY spectra of two non-selfcomplementary undecanucleotide duplexes. From the observed NOEs we do not detect any significant distortion of the helix when a G-C pair is replaced by a G-T pair and the normal interresidue connectivities can be followed through the mismatch site. We conclude that the 2D spectra of the non-exchangeable protons do not allow differentiation between a wobble or rare tautomer form for the mismatch. NOE measurements in H2O, however, clearly show that the mismatch adopts a wobble structure and give information on the hydration in the minor groove for the G-T base pair which is embedded between two A-T base pairs in the sequence.

Adenosine↗

PubMed1987

dam methylase from E. coli. Circular dichroism investigations of the secondary structure and influence of S-adenosylmethionine.

The enzyme dam methylase which recognizes and methylates the adenine in the palindromic sequence GATC in DNA was isolated and the secondary structure was determined by CD spectroscopy and various predicting methods from the amino acid sequence. The interaction of dam methylase with S-adenosylmethionine was studied by CD spectroscopy indicating a decrease of the percentage of alpha-helix as the amount of S-adenosylmethionine bound to the enzyme was increased.

Circular Dichroism↗

PubMed1987

NMR and CD studies on an oligonucleotide containing N4-methylcytosine.

The hexamer d(CGm4CGCG) exists predominantly as a right handed B form helix at 20 degrees C in 150 mM NaCl, as shown by 2D NOE spectra. Under these conditions a minor species is also observed which corresponds to the single strand in slow exchange on a proton NMR time scale with the double strand. This exchange is unusually slow and separate resonances for the two species are seen up to 65 degrees C. At 50 degrees C the lifetime of the single strand species is 0.85 s. Under high salt conditions the hexamer is partly converted into the Z form, but the complete transition is only observed at 5M NaCl at -6 degrees C.

Circular Dichroism↗

PubMed1986

Structures of mismatched base pairs in DNA and their recognition by the Escherichia coli mismatch repair system.

The Escherichia coli mismatch repair system does not recognize and/or repair all mismatched base pairs with equal efficiency: whereas transition mismatches (G X T and A X C) are well repaired, the repair of some transversion mismatches (e.g. A X G or C X T) appears to depend on their position in heteroduplex DNA of phage lambda. Undecamers were synthesized and annealed to form heteroduplexes with a single base-pair mismatch in the centre and with the five base pairs flanking each side corresponding to either repaired or unrepaired heteroduplexes of lambda DNA. Nuclear magnetic resonance (n.m.r.) studies show that a G X A mismatch gives rise to an equilibrium between fully helical and a looped-out structure. In the unrepaired G X A mismatch duplex the latter predominates, while the helical structure is predominant in the case of repaired G X A and G X T mismatches. It appears that the E. coli mismatch repair enzymes recognize and repair intrahelical mismatched bases, but not the extrahelical bases in the looped-out structures.

Bacteriophage lambda↗

PubMed1986

The low ionic strength form of the sodium salt of poly(dm5C-dG) is a B DNA.

The low-salt Z form of poly(dm5C-dG) reported in the literature was shown to be due to minute amounts of bivalent cations. If the polymer was purified by dialysis against 1 mM EDTA, poly(dm5C-dG) showed the normal B form. No change in this B form was observed up to 0.8 M NaCl. Very small amounts of Mg2+ or Ca2+ (10(-6) to 10(-5) M) induced the characteristic Z form CD spectrum, which could easily and rapidly be reverted to B-DNA by addition of EDTA.

Circular Dichroism↗

PubMed1985

Consequences of methylation on the amino group of adenine. A proton two-dimensional NMR study of d(GGATATCC) and d(GGm6ATATCC).

A two-dimensional 500-MHz 1H-NMR study of two oligonucleotides, d(GGATATCC) and d(GGm6ATATCC), is presented in which we have investigated the effects of adenine methylation. The two-dimensional nuclear Overhauser spectra (NOESY) show that both oligonucleotides adopt a normal right-handed B-type helix and one-dimensional nuclear Overhauser enhancement (NOE) studies demonstrate that any difference in conformation must be small. However methylation drastically slows down the helix in equilibrium coil exchange which becomes slow on a proton NMR time scale. While d(GGATATCC) fits a two-site exchange model, d(GGm6ATATCC) does not and we invoke the presence of a third species which may be an intermediate in helix formation. NMR and ultraviolet spectroscopy show that methylation destabilizes the helix, measured by the melting temperature and enthalpy of dissociation.

Adenine↗

PubMed1985

NMR studies on oligodeoxyribonucleotides containing the dam methylation site GATC. Comparison between d(GGATCC) and d(GGm6ATCC).

The conformation of two hexanucleotides, d(GGATCC) and d(GGm6ATCC), has been studied by proton nuclear magnetic resonance. Nuclear Overhauser effect (NOE) measurements on d(GGATCC) are in agreement with a normal B form right-handed helical structure. The single- and double-strand resonances are in fast exchange on a proton NMR time scale. The exchange is observed to be slow for d(GGm6ATCC); up to the Tm, separate resonances are observed for each state, though above the Tm exchange becomes more rapid. The preferred orientation of the adenosine methylamino group (methyl cis to N1) hinders base-pair formation. At 0 degree C irradiation of the m6A-T imino proton gives an NOE to AH2, showing that base pairing is Watson-Crick. Intra- and interresidue NOEs show that the helix is right handed and in the B form. Comparing results on the two oligomers demonstrates that adenosine methylation induces little or no change in the conformation of the helix but reduces the Tm from 45 to 32 degrees C. All of the amino proton resonances, as well as the imino resonances, have been assigned. From NOE experiments on the unmethylated oligomer we have located the Watson-Crick and non-Watson-Crick adenosine amino protons. At 0 degree C these resonances show broadening due to rotation of the amino group, and their rotation is slightly slower than for the adjacent guanosine amino group, though both these amino groups have lifetimes of less than 10 ms at 0 degree C. The imino protons show normal behavior, disappearing from the spectra ca. 20 degrees C below the Tm.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed1985

A----Z transition in the synthetic hexanucleotide (dCdGfl)3.

500 MHz proton NMR and NOE measurements on (dCdGfl)3 show that at very low ionic strength the hexanucleotide adopts an A-DNA conformation, whereas at high salt concentrations a Z-form is found. At intermediate salt concentrations the two species are in slow exchange on the proton NMR time scale. This transition was also observed by characteristic changes in the CD spectra.

Circular Dichroism↗

PubMed1985

2'-Fluoro-2'-deoxycytidine triphosphate as a substrate for RNA- and DNA-dependent DNA polymerases.

The ability of the analog 2'-fluoro-2'-deoxycytidine triphosphate (dCflTP) to be used as a substrate in the reactions catalyzed by Xenopus laevis oocytes DNA polymerase alpha and AMV reverse transcriptase has been studied. The apparent Km values for dCTP and dCflTP, using activated DNA as templates, were 0.6 microM and 7 mM with DNA polymerase alpha and 0.14 microM and 7 microM with AMV reverse transcriptase, respectively. As observed with dCTP, aphidicolin was a noncompetitive inhibitor in the DNA polymerase alpha-catalyzed DNA synthesis; the Ki values were about 2 microM for both substrates. dCflTP can also be incorporated into DNA synthetized by other eukaryotic DNA polymerases and by reverse transcriptase with RNA as a template, both in the presence or absence of (dT)12 primer.

Animals↗

PubMed1985

2'-Fluoro-2'-deoxypolynucleotides as templates and inhibitors for RNA- and DNA-dependent DNA polymerases.

Poly(2'-fluoro-2'-deoxyadenylic acid) (poly(dAfl)) and poly(2'-deoxycytidylic acid) (poly(dCfl)) were tested as templates in DNA synthesis reactions catalyzed by Xenopus laevis oocytes DNA polymerase alpha, mouse cell DNA polymerase gamma and avian myeloblastis virus (AMV) reverse transcriptase. Poly(dAfl).(dT)12 can fully substitute for poly(rA).(dT)12 as template with DNA polymerase gamma, to 50% with reverse transcriptase, but was poorly recognized by DNA polymerase alpha. DNA synthesis by reverse transcriptase with poly(dCfl).(dG)12 as template was 50% of that with poly(rC).(dG).(dG)12. The use of 2'-fluoropolymers as templates was more efficient at 37 degrees C than at 25 degrees C. No appreciable differences on the fidelity of DNA synthesis by reverse transcriptase were observed when dCMP misincorporation was measured with poly(dAfl).(dT)12 or poly(rA).(dT)12 as template primers. Poly(C) and poly-2'-O-methylcytidylic acid had no significant effect on the reaction catalyzed by DNA polymerase gamma and reverse transcriptase, independent of the synthetic polynucleotide complex utilized as template. On the other hand, poly(dCfl) was an inhibitor when poly(rA).(dT)12 or poly(dA).(dT)12 were used as templates, but not when poly(dAfl).(dT)12 was employed. Analogous results have been obtained with activated DNA and AMV 70 S RNA as templates in the reverse transcriptase reaction. The inhibition by poly(dCfl) was noncompetitive with regard to TTP, poly(dA) and poly(rA). Xenopus laevis oocytes DNA polymerase alpha was not inhibited by poly(dCfl).

Animals↗