Solution structure of a DNA three-way junction containing two unpaired thymidine bases. Identification of sequence features that decide conformer selection.

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.

Mutational and structural analyses of the ribonucleotide reductase inhibitor Sml1 define its Rnr1 interaction domain whose inactivation allows suppression of mec1 and rad53 lethality.

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.

Conformation of the circular dumbbell d: structure determination and molecular dynamics.

The circular DNA decamer 5'-d-3' was studied in solution by means of NMR spectroscopy and molecular dynamics in H2O. At a temperature of 269 K, a 50/50 mixture of two dumbbell structures (denoted L2L2 and L2L4) is present. The L2L2 form contains three Watson-Crick C-G base pairs and two two-residue loops is opposite parts of the molecule. On raising the temperature from 269 K to 314 K, the L2L4 conformer becomes increasingly dominant (95% at 314 K). This conformer has a partially disrupted G(anti)-C(syn) closing base pair in the 5'-GTTC-3' loop with only one remaining (solvent-accessible) hydrogen bond between NH alpha of the cytosine dC(1) and O6 of the guanine dG(8). The opposite 5'-CTTG-3' loop remains stable. The two conformers occur in slow equilibrium (rate constant 2-20 s-1). Structure determination of the L2L2 and L2L4 forms was performed with the aid of a full relaxation matrix approach (IRMA) in combination with restrained MD. Torsional information was obtained from coupling constants. Coupling constant analysis (3JHH, 3JHP, 3JCP) gave detailed information about the local geometry around backbone torsion angles beta, gamma, delta, and epsilon, revealing a relatively high flexibility of the 5'-GTTC-3' loop. The values of the coupling constants are virtually temperature-independent. 'Weakly constrained' molecular dynamics in solvent was used to sample the conformational space of the dumbbell. The relaxation matrices from the MD simulation were averaged over to predict dynamic NOE volumes. In order to account for the 1:1 conformational mixture of L2L2 and L2L4 present at 271 K, we also included S2 factors and averaging of the -averaged relaxation matrices. On matrix averaging, the agreement of NOE volumes with experiment improved significantly for protons located in the thermodynamically less stable 5'-GTTC-3' loop. The difference in stability of the 5'-CTTG-3' and 5'-GTTC-3' loops is mainly caused by differences in the number of potential hydrogen bonds in the minor groove and differences in stacking overlap of the base pairs closing the minihairpin loops. The syn conformation for dC(1), favored at high temperature, is stabilized by solvation in the major groove. However, the conformational properties of the dC(1) base, as deduced from R-factor analysis and MD simulations, include a large flexibility about torsion angle chi.

Slow conformational exchange in DNA minihairpin loops: a conformational study of the circular dumbbell d.

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-3' (I) is studied in solution by means of nmr spectroscopy. At a temperature of 269 K the molecule occurs in a 50/50 mixture of two dumbbell structures (denoted L2L2 and L2L4). L2L2 contains three Watson-Crick C-G base pairs and two two-residue loops (H2-family type) in opposite parts of the molecule. On raising the temperature from 269 to 314 K, the L2L4 conformer becomes increasingly dominant (95% at 314 K). This conformer has a partially disrupted closing G-C base pair in the 5'-GTTC-3' loop with only one remaining solvent-accessible hydrogen bond between NH alpha of the cytosine C(1) and O6 of the guanine G(8), whereas the opposite 5'-CTTG-3' loop remains stable. The disruption of the C(1)-G(8) base pair in the L2L4 form is correlated with the presence of a syn orientation for the C(1) base at the 5'-3' loop-stem junction in the 5'-GTTC-3' loop. The two conformers, L2L2 and L2L4, occur in slow equilibrium (2-20 s-1). Moderate line broadening of specific 1H, 13C, and 31P resonances of residues C(1), G(8), T(9), and T(10) at low temperatures, due to chemical exchange between L2L2 and L2L4, show that the interconversion from an anti to syn conformer in residue C(1) has a small local effect on the structure of the dumbbell. T1 relaxation measurements, chemical-shift considerations, and complete band-shape calculations of the exchange process of the G(8) imino proton reveal a possibility for the existence of multiconformational states in the anti-syn equilibrium.

Thermodynamics of melting of the circular dumbbell d.

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-3' (I) and reference compounds 5'-d-3' (II) and 5'-d(GCG-TC-CGC)-3' (III) are studied by means of nmr spectroscopy. Molecules I and II adopt dumbbell structures closed on both sides by a two-membered hairpin loop. At low temperature I consists of a mixture of two slowly exchanging forms, denoted L2L2 and L2L4. The low-temperature L2L2 form is the fully intact minihairpin structure with three Watson-Crick C-G base pairs. The high-temperature form, L2L4, contains a partially disrupted closing G-C base pair in the 5'-GTTC-3' loop, with the cytosine base placed in a syn orientation. The opposite 5'-CTTG-3' loop remains stable. A study of the noncircular hairpin structure III shows similar conformational behavior for the 5'-GTTC-3' loop as found in I; a syn orientation for C(6) and two slowly exchanging imino proton signals for G(3). The melting point Tm of II was estimated to lie above 365 K. The Tm value of the duplex stem and the 5'-CTTG-3' loop of the L2L4 form of I is 352 +/- 2 K. The delta H0 is calculated as -89 +/- 10 kJ/mol. The Tm value determined for the individual residues of the 5'-GTTC-3' loop lies 4 degrees-11 degrees lower. The enthalpy delta H0 of melting the thymine residues in the 5'-GTTC-3' loop is calculated to be -61 +/- 7 kJ/mol. Thermodynamic data of the equilibrium between the slowly exchanging two- and four-membered loop conformers of I reveal an upper limit for delta H0 of +30 kJ/mol in going from a two-membered to a four-membered loop, in agreement with the enthalpy difference of +28 kJ/mol between the two loops at the Tm midpoint. For hairpin III the upper limit for delta H0 in going from a two-membered to a four-membered loop amounts to +/- 21 kJ/mol.(ABSTRACT TRUNCATED AT 400 WORDS)

Heteronuclear NMR of DNA with the heteronucleus in natural abundance: facilitated assignment and extraction of coupling constants.

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.

An NMR study of the conformation and thermodynamics of the circular dumbbell d [formula: see text] Slow exchange between two- and four-membered hairpin loops.

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.