Subject(s)
Acer , Ascomycota/metabolism , Horse Diseases/diagnosis , Multiple Acyl Coenzyme A Dehydrogenase Deficiency/veterinary , Acer/microbiology , Animal Feed/analysis , Animals , Ascomycota/growth & development , Diet/veterinary , Fatal Outcome , Horse Diseases/blood , Horse Diseases/etiology , Horses , Hypoglycins/metabolism , Magnetic Resonance Spectroscopy , Male , Multiple Acyl Coenzyme A Dehydrogenase Deficiency/blood , Multiple Acyl Coenzyme A Dehydrogenase Deficiency/diagnosis , Multiple Acyl Coenzyme A Dehydrogenase Deficiency/etiology , Netherlands , Plant Leaves/metabolism , Plant Leaves/microbiology , Seeds/metabolismABSTRACT
The solution structure of a DNA three-way junction (3H) containing two unpaired thymidine bases at the branch site (3HT2), was determined by NMR. Arms A and B of the 3HT2 form a quasi-continuous stacked helix, which is underwound at the junction and has an increased helical rise. The unstacked arm C forms an acute angle of approximately 55 degrees with the unique arm A. The stacking of the unpaired thymidine bases on arm C resembles the folding of hairpin loops. From this data, combined with the reported stacking behavior of 23 other 3HS2 s, two rules are derived that together correctly reproduce their stacking preference. These rules predict, from the sequence of any 3HS2, its stacking preference. The structure also suggests a plausible mechanism for structure-specific recognition of branched nucleic acids by proteins.
Subject(s)
Base Pairing , DNA/chemistry , DNA/genetics , Thymidine/metabolism , Base Sequence , DNA/metabolism , High Mobility Group Proteins/metabolism , Hydrogen Bonding , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Sequence Data , Protons , RNA, Catalytic/chemistry , RNA, Catalytic/genetics , RNA, Catalytic/metabolism , Recombination, Genetic/genetics , Solutions , Substrate Specificity , Thymidine/geneticsABSTRACT
In budding yeast, MEC1 and RAD53 are essential for cell growth. Previously we reported that mec1 or rad53 lethality is suppressed by removal of Sml1, a protein that binds to the large subunit of ribonucleotide reductase (Rnr1) and inhibits RNR activity. To understand further the relationship between this suppression and the Sml1-Rnr1 interaction, we randomly mutagenized the SML1 open reading frame. Seven mutations were identified that did not affect protein expression levels but relieved mec1 and rad53 inviability. Interestingly, all seven mutations abolish the Sml1 interaction with Rnr1, suggesting that this interaction causes the lethality observed in mec1 and rad53 strains. The mutant residues all cluster within the 33 C-terminal amino acids of the 104-amino-acid-long Sml1 protein. Four of these residues reside within an alpha-helical structure that was revealed by nuclear magnetic resonance studies. Moreover, deletions encompassing the N-terminal half of Sml1 do not interfere with its RNR inhibitory activity. Finally, the seven sml1 mutations also disrupt the interaction with yeast Rnr3 and human R1, suggesting a conserved binding mechanism between Sml1 and the large subunit of RNR from different species.
Subject(s)
Cell Cycle Proteins , Enzyme Inhibitors , Fungal Proteins/genetics , Protein Kinases/genetics , Protein Serine-Threonine Kinases , Ribonucleotide Reductases/antagonists & inhibitors , Saccharomyces cerevisiae Proteins , Suppression, Genetic , Checkpoint Kinase 2 , Chromosomes, Fungal , DNA Mutational Analysis , Humans , Intracellular Signaling Peptides and Proteins , Mutation, Missense , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Solutions , Species Specificity , Trefoil Factor-2 , Two-Hybrid System TechniquesSubject(s)
Homeodomain Proteins/chemistry , Magnetic Resonance Spectroscopy/methods , Nerve Tissue Proteins , Animals , Binding Sites , Carbon Isotopes , Homeodomain Proteins/metabolism , Hydrogen/chemistry , In Vitro Techniques , LIM-Homeodomain Proteins , Nitrogen Isotopes , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Rats , Transcription FactorsABSTRACT
In recent years various examples of highly stable two-residue hairpin loops (miniloops) in DNA have been encountered. As the detailed structure and stability of miniloops appear to be determined not only by the nature and sequence of the two bases in the loop, but also by the closing base pair, it is desirable to carry out in-depth studies of especially designed small model DNA compounds. Therefore, a circular DNA dumbbell-like molecule is tailored to consist of a stem of three Watson-Crick base pairs, flanked on each side by a minihairpin loop. The resulting circular DNA decamer 5'-d
Subject(s)
DNA, Circular/chemistry , Nucleic Acid Conformation , Base Sequence , Kinetics , Molecular Sequence Data , ThermodynamicsABSTRACT
The conformational behavior of DNA minihairpin loops is sensitive to the directionality of the base pair that closes the loop. Especially tailored circular dumbbells, consisting of a stem of three Watson-Crick base pairs capped on each side with a minihairpin loop, serve as excellent model compounds by means of which deeper insight is gained into the relative stability and melting properties of hairpin loops that differ only in directionality of the closing pair: C-G vs G-C. For this reason the thermodynamic properties of the circular DNA decamers 5'-d
Subject(s)
DNA, Circular/chemistry , Nucleic Acid Conformation , Base Sequence , Chemical Phenomena , Chemistry, Physical , Molecular Sequence Data , ThermodynamicsABSTRACT
The circular DNA decamer 5'-d
Subject(s)
DNA, Circular/chemistry , Magnetic Resonance Spectroscopy , Models, Molecular , Nucleic Acid Conformation , ProtonsABSTRACT
Two heteronuclear proton-carbon NMR experiments are applied to the DNA-octamer d(TTGGCCAA)2 with carbon in natural abundance. They lead to a complete assignment of the carbon resonances of the sugars and bases. In addition, several heteronuclear coupling constants, proton-carbon as well as proton-phosphorous and phosphorous-carbon, were determined. The information can be obtained in a reasonable measuring time and offers valuable information for a detailed picture of DNA structure.
Subject(s)
DNA/chemistry , Nucleic Acid Conformation , Oligodeoxyribonucleotides/chemistry , Base Sequence , Magnetic Resonance Spectroscopy/methodsABSTRACT
The circular DNA decamer 5'-d [formula: see text] 3' is studied in solution by means of NMR spectroscopy. At low temperature the molecule adopts a dumbbell structure with three Watson-Crick C-G base pairs and two two-residue loops in opposite parts of the molecule. On raising the temperature another conformer appears, in which the closing C-G base pair in the 5'-GTTC-3' loop is disrupted, whereas the opposite 5'-CTTG-3' loop remains stable. The two conformers are in slow equilibrium over a limited temperature range.