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Biomedical subjects

G Krauss

Publications and source records attributed to G Krauss.

At least 55 records · Page 3Linked to original sources
PubMed1985

Analysis of secondary structures in M13mp8 (+) single-stranded DNA by the pausing of DNA polymerase alpha.

The pausing of DNA replication has been used as a tool for analyzing secondary structures in a single-stranded DNA. M13mp8 (+) single-stranded DNA was replicated in vitro by the DNA polymerase alpha from calf thymus. The positions of pausing were determined from DNA sequencing gels. All experimentally observed pausing sites could be correlated with computer-predicted secondary structures of the M13 single-stranded DNA. In the computer calculations of the secondary structures, long-range base-pairing, G.T mispairs and loop-out of bases were allowed. By using six different primers, the pausing site pattern and the corresponding secondary structure map of a region comprising 1400 nucleotides of the M13 genome has been established. Our experiments indicate that the M13 DNA is highly structured. Most of the stable structures are clustered around the origin of replication. With fragments of the M13 DNA, we show that long-range base-pairing exists in the M13 single-stranded genome and we present evidence for tertiary structure interactions. Furthermore we observe structures that form newly during the course of replication. The Escherichia coli single-stranded DNA-binding protein facilitates replication through the barriers.

Animals↗
PubMed1985

On the fidelity of DNA polymerase alpha: the influence of alpha-thio dNTPs, Mn2+ and mismatch repair.

The phi X174am16 revertant system has been used to investigate the influence of alpha-thio-dNTPs and of Mn2+ on the fidelity of the 9S DNA polymerase alpha from calf thymus. Upon substituting dGTP by alpha-thio-dGTP during the in vitro replication, a nearly tenfold decrease in the frequency of G:G and G:T mispairs is observed. The formation of all other mispairs is not changed in the presence of the corresponding alpha-thio-dNTP. Mn2+ at concentrations of 0.5 mM does not influence the frequencies of the mispairs. The expression rate of errors formed during in vitro replication in the (-) strand has been determined for all mispairs detectable in the phi Xam16 system. The (-) strand expression of G:T, T:T and C:T mismatches is about 50%, whereas for A:G, G:G and C:A mismatches it is clearly below 50%. We conclude that the different base-base mismatches are repaired with different efficiencies.

Animals↗
PubMed1985

The primase activity of DNA polymerase alpha from calf thymus.

The nearly homogeneous 9 S DNA polymerase alpha from calf thymus contains a primase activity that allows priming of DNA synthesis on single-stranded templates in the presence of ribonucleoside triphosphates. Both on synthetic and natural single-stranded templates, RNA primers of 8-15 nucleotides in length are formed. In the absence of dNTPs, primers of some hundred nucleotides in length are observable. ATP and/or GTP are required for the priming reaction. UTP and CTP cannot initiate the RNA synthesis. M13 single-stranded DNA can be converted to the nicked double helical form upon primase-primed replication by the 9 S enzyme. Priming occurs mostly at specific sites on the M13 genome and replication products of up to 6000 nucleotides in length are formed. In the presence of the single-stranded DNA binding protein from Escherichia coli, specificity of priming is strongly increased. The primase is inhibited by salt and actinomycin; it is insensitive to alpha-amanitin and N-ethylmaleimide. Due to the strong complex formation between DNA polymerase and primase, it has not been possible to separate the two activities of the multisubunit 9 S enzyme.

Adenosine Triphosphate↗
PubMed1984

Escherichia coli single-stranded DNA binding protein is mobile on DNA: 1H NMR study of its interaction with oligo- and polynucleotides.

The interaction of the Escherichia coli single-stranded DNA binding protein (SSB) with oligo- and poly-nucleotides has been studied by 270-MHz 1H NMR spectroscopy and fast kinetic techniques. d(pT)8 and poly(dT) were used to study noncooperative and cooperative binding, respectively. The H6, H1', and CH3 resonances of d(pT)8 are high-field shifted by less than 0.05 ppm, and H8 and H2 of poly(dA) are low-field shifted upon complexation. The protein resonances remain virtually unshifted. The small shifts upon complexation provide no evidence for extensive stacking interactions between the nucleotide bases and aromatic amino acid side chains of SSB. The d(pT)8 and poly(dA) signals are broadened to about 30 Hz whereas the resonances of poly(dT) are broadened beyond detection upon stoichiometric complexation. Continuous broadening of all poly(dT) signals even at a 10-fold excess of poly(dT) indicates fast exchange of SSB between different binding sites. Dissociation and reassociation rates determined from stopped-flow experiments are too slow by at least 2 orders of magnitude to account for the experimental line widths. Therefore, we conclude that SSB translocates without dissociation from the DNA template. A model for the translocation is outlined. It is based on partial dissociation of octamer sections of poly(dT) from the complex with a rate constant as previously published for the dissociation of d(pT)8 from SSB.

DNA-Binding Proteins↗
PubMed1984

Replication of M13mp7 single-stranded DNA in vitro by the 9-S DNA polymerase alpha from calf thymus.

The replication of M13 single-stranded DNA by the 9S DNA polymerase alpha from calf thymus has been studied in vitro. Priming conditions, the nature of the replication products and conditions for optimal elongation have been investigated. Oligonucleotides comprising only four nucleotides can serve as primers. Both ribo and deoxy oligonucleotides can be elongated. Priming by the short oligonucleotides occurs at multiple sites on the M13 genome. If replication is primed at single sites with a specific pentadecamer or with RNA in the origin of replication, specific pausing sites are observed. These pausing sites can partly be correlated with secondary structures in the template DNA. Addition of Escherichia coli single-stranded DNA binding protein leads to a weakening of pausing sites and to the synthesis of longer products. The 9S enzyme is able to proceed through most of the pausing sites resulting in the synthesis of product molecules as long as 6600 nucleotides. The 9S DNA polymerase alpha contains a potent DNA primase activity which enables it to initiate replication on a single-stranded template in the presence of the four NTPs . However, priming is also possible in the presence of ATP alone. The priming sites are not randomly distributed over the M13 DNA.

Animals↗
PubMed1983

The elongation of mismatched primers by DNA polymerase alpha from calf thymus.

The ability of the 9S and 5.7S DNA polymerase alpha subspecies from calf thymus in elongating a mismatched primer terminus has been investigated. With poly(dA) as template, the elongation rate for (dT)8dG, (dT)8dC and (dT)10dGdT is 20-fold lower for the 9S enzyme and 5-fold lower for the 5.7S enzyme as compared to (dT)10. The presence of a second mismatch at the primer terminus reduces the elongation rate further by a factor of two. Exonucleolytic excision of the mismatches can be excluded. With (dT)8dG (dT)n as primer we show, that at least five T-residues have to follow the mismatch in order to establish the elongation rate of a perfectly paired primer. The KM value for (dT)10 dG as primer is 400 nM as compared to 10 nM for (dT)10. Addition of Mn2+ increases the relative efficiency of elongation of the mismatched primers.

Animals↗
PubMed1983

Subspecies of DNA polymerase alpha from calf thymus with different fidelity in copying synthetic template-primers.

Three different subspecies of DNA polymerase alpha from calf thymus sedimenting at 9 S, 7 S and 5.7 S have been investigated with respect to their accuracy of in vitro DNA synthesis on poly (dA) (dT)16 and poly d(AT) as template-primers. Our results indicate that the structure of DNA polymerase alpha has a strong influence on the accuracy of DNA synthesis. The 9 S enzyme shows a misincorporation frequency of about 1:100 000. An error rate of 1:15 000 is measured for the 7 S species. The 5.7 S enzyme for which an error rate of 1:3 000 is determined, has to be considered as error prone. Lowering the rate of DNA synthesis leads to a decrease in fidelity. The single stranded DNA binding protein from E.coli increases the accuracy of the 5.7 S and the 7 S enzyme by a factor of two. Mn2+ decreases the fidelity of all three subspecies in a concentration dependent manner.

Animals↗
PubMed1983

Accuracy of DNA polymerase-alpha in copying natural DNA.

The fidelity of DNA polymerase-alpha from calf thymus (9S enzyme) in copying bacteriophage phi174am16 DNA in vitro has been determined from the frequency of production of different revertants. In the self-priming reaction we were able to measure the frequencies of base pairing mismatches during the course of replication on biasing the ratios of deoxynucleoside triphosphates. The frequency of dGTP:T, dGTP:G and dATP:G mismatches were 7.6 x 10(-5), 4.4 x 10(-5) and 2.8 x 10(-5), respectively, at equal concentrations of the deoxynucleoside triphosphates. dCTP:A, dGTP:A, dCTP:T and dTTP:T mismatches were below the limit of detection (<5 x 10(-6)). A synthetic dodecamer primer with a 3' end covering the first two bases of the amber codon was used to determine the misinsertion frequency of the first nucleotide incorporated. This gave a misinsertion frequency of 1.5 x 10(-4) for the dGTP:T mismatch, which is slightly higher than that observed from the pool bias studies. Further, it showed no sensitivity to biasing the nucleotide pool, suggesting a different mechanism for the incorporation of the first nucleotide. These data do not support 'energy-relay'-like models for achieving high accuracy in eukaryotes. The observed misinsertion frequencies were corrected for mismatch repair of the heteroduplexes during the transfection experiments by parallel experiments using a mismatched primer. This was synthesized to have the same G:T mismatch as produced in the preceding experiment.

Animals↗
PubMed1981

Purification of a 9S DNA polymerase alpha species from calf thymus.

a DNA polymerase alpha species from calf thymus has been purified 12 000-fold to near homogeneity. The enzyme sediments under high salt conditions in the preparative ultracentrifuge as a homogeneous band at 9S. The specific activity is 50 000-70 000 units/mg of protein. Polypeptides of 148 000, 59 000, and 48 000 daltons are detectable. The molecular weight as estimated from gradient gel electrophoresis is about 500 000. The 9S DNA polymerase is free from terminal nucleotidyl transferase activity and does not exhibit endonuclease or exonuclease activity. It is inhibited by low concentrations of salt, aphidicolin, and N-ethylmaleimide.

Animals↗
PubMed1981

Escherichia coli single-strand deoxyribonucleic acid binding protein: stability, specificity, and kinetics of complexes with oligonucleotides and deoxyribonucleic acid.

The complex formation between the single-strand DNA binding protein (ssB protein) from Escherichia coli and oligonucleotides and single-stranded DNA has been studied by using fluorescence titrations, ultracentrifugation measurements, and fast kinetic techniques. Determination of the stoichiometries of oligo(dT)--ssB complexes shows that each of the four subunits of the ssB protein represents a binding site for an oligonucleotide about eight residues long. Occupation of all four binding sites with oligo(dT) or poly(dT) leads to 80% quenching of the intrinsic protein fluorescence. The binding sites are nearly equivalent and independent. For d(pT)16, the intrinsic binding constant is 6 X 10(5) M-1, and for d(pT)30-40, which is long enough to extend continuously over the ssB tetramer, the binding constant is higher than 5 X 10(8) M-1. Oligoadenylates bind about 2 orders of magnitude weaker than the corresponding oligo(dT) species. The binding of oligo(dT) is very weakly dependent on ionic strength, in contrast to the oligo(dA)--ssB complex formation. For d(pT)8, d(pT)16, and d(pT)30-40, the complex formation can be described by a simple one-step reaction. The strength of the interaction is mainly expressed in the rate constant of dissociation. In the cooperative complexes with poly(dT) or poly(dA), all four binding sites on the ssB tetramer are also occupied. It is concluded that single-stranded DNA is coiled around the ssB molecule. Fluorescence melting experiments of the complexes show that the conformation of the single-stranded DNA has a strong influence on the stability of the complexes.

Binding Sites↗
PubMed1980

Purification and partial characterization of a DNA polymerase alpha species from calf thymus.

We have purified a DNA polymerase alpha species from calf thymus to near homogeneity. The enzyme sediments at 5.7 S and contains two polypeptides of 123000 and 134000 daltons in about equimolar ratio. The enzyme is inhibited by aphidicolin and N-ethylmaleimide, and retains its activity in buffers containing moderate salt conditions. Activated DNA is a better substrate than poly-(dA) . (dT) 10.

Ampholyte Mixtures↗
PubMed1980

Survival and mutagenesis in UV-irradiated phage: multi-hit kinetics of mutation induction and lack of indirect induction by infection with UV-irradiated phage of error-prone repair.

The paper is concerned with the question of whether WEIGLE-reactivation (WR) and WEIGLE-mutagenesis (WM) can be indirectly induced by infection with UV-irradiated phage. Experiments neither with phage lambda of Escherichia coli nor with phage chi of Serratia marcescens show such induction-In this respects phage DNA differs from F'-DNA or Hfr-DNA; possible explanations are discussed. In both systems clear phage mutations can also be induced by UV without irradiation of the host cells; they appear, in unirradiated and irradiated host cells, with an increase of frequency which is greater than proportional to the UV dose. It is concluded that mutation induction of phage in the unirradiated host cells is due to a low level constitutive mutagenic repair; this could either be due to "spontaneous" induction of the mutagenic SOS-function or it could be a mechanism different from this one. Host irradiation would give rise to additional activity by the induced SOS-function leading to WR and WM. It is further concluded that deviation of the induction kinetics from a linear dose-dependence is not due to the necessary induction of SOS-functions.

Bacteriophage lambda↗
PubMed1980

[Growth inhibition by phenylalanine and tyrosine--metabolism of phenylalanine in Flavobacterium devorans (author's transl)].

Growth of a chloramphenicol-resistant mutant of Flavobacterium devorans (F. devorans r) was inhibited by L-phenylalanine and L-tyrosine and furthermore by L-tryptophan, 4-amino-benzoate, anthranilate and homogentisate. The inhibitionwas reversed partially by simultaneous addition of aromatic amino acids and 4-amino-benzoate. After cultivating F. devorans r in a glucose-minimal medium containing L-phenylalanine, the metabolites L-tyrosine, 4-hydroxyphenylpyruvate, 4-hydroxyphenylacetate, 4-hydroxybenzaldehyd, 4-hydroxybenzoate, homogentisate and anthranilate were found. Growth inhibition and a hypothetical degrading pathway of phenylalanine are discussed.

Aminobenzoates↗
PubMed1980

[A prephenate dehydratase from Flavobacterium devorans stimulated by aromatic amino acids (author's transl)].

Isolation and properties of prephenate dehydratase from Flavobacterium devorans r are described. The enzyme was enriched 43-fold by ammonium sulfate precipitation, by gel chromatography on ultrogel ACA-34 and by hydrophobic chromatography on decylagarose. The molecular weight was estimated to be 1.35 x 10(5). The enzyme was activated in 1mM solutions of L-tyrosine 4,4-fold, of L-phenylalanine 3.9fold and by L-tryptophan 1.9-fold in 0.02M Tris/HCl buffer at pH 7.7. The Km values were, when adding the activator, at L-tyrosine 1.5 x 10(-5) M, at L-phenylalanine 1.3 x 10(-5) M and at L-tryptophan 1 x 10(-5) M. Regulation of aromatic amino acid is discussed in connection with the prephenate dehydratase.

Chorismate Mutase↗
PubMed1979

Conformation transitions of a tRNA--aminoacyl-tRNA synthetase complex induced by tRNAs bearing different modifications in the 3' terminus.

The influence of modifications of the 3'-terminal adenosine of tRNAPhe (yeast) on the complex formation between this tRNA and phenylalanyl-tRNA synthetase (yeast) has been investigated by using fluorescence titrations and fast kinetic techniques. Subtle changes in the 3' terminus are reflected by distinct alterations in the two-step recognition process which had been demonstrated earlier for the native substrate tRNAPheCCA [Krauss, G., Riesner, D., & Maass, G. (1977) Nucleic Acids Res. 4, 2253--2262]. Binding experiments with tRNAPheCC, tRNAPheCCA-ox-red, tRNAPheCC2'dA, tRNAPheCC3'dA, tRNAPheCC-formycin, and tRNAPheCC-formycin-ox-red confirm that the 3'-terminal adenosine participates in a conformational change of the tRNA--synthetase complex. This is valid in both the absence and presence of phenylalaninyl-5'-AMP, the alkyl analogue of the aminoacyladenylate. As compared to tRNAPheCCA, a slower conformational change is observed with the competitive inhibitor tRNAPheCC-formycin-ox-red. The reaction enthalpy and/or the quench of the Y-base fluorescence that accompany the conformational change are altered upon binding of tRNAPheC2'dA, tRNAPheCC3'dA, and tRNAPheCC-formycin. It is evident that the final adaptation between tRNA and its synthetase in the complex is determined by the chemical nature of the 3'-terminal nucleotide. This is of vital importance for the specificity of the aminoacylation process.

Amino Acyl-tRNA Synthetases↗
PubMed1979

The binding of tyrosinyl-5'-AMP to tyrosyl-tRNA synthetase (E.coli).

The binding between tyrosyl-tRNA synthetase (E.coli) and the alkylanalogue of the aminoacyladenylate, tyrosinyl-5'-AMP, has been investigated by fluorescence titrations and rapid mixing experiments. Tyrosyl-tRNA synthetase has two equivalent and independent binding sites for tyrosinyl-5'-AMP. The intrinsic binding constant is 4 x 10(7)M-1. The binding sites for tRNATyr and tyrosinyl-5'-AMP are independent of each other, the anticooperative mode of tRNA binding being preserved in the presence of tyrosinyl-5-AMP.

Adenosine Monophosphate↗