Nuclear Overhauser effect studies of the conformations and binding site environments of deoxynucleoside triphosphate substrates bound to DNA polymerase I and its large fragment

The conformations and binding site environments of Mg2+TTP and Mg2+dATP bound to Escherichia coli DNA polymerase I and its large (Klenow) fragment have been investigated by proton NMR. The effect of the large fragment of Pol I on the NMR line widths of the protons of Mg2+TTP detected one binding site for this substrate with a dissociation constant of 300 +/- 100 microM and established simple competitive binding of deoxynucleoside triphosphates at this site in accord with previous equilibrium dialysis experiments with whole Pol I [Englund, P. T., Huberman, J.A., Jovin, T.M., & Kornberg, A. (1969) J. Biol. Chem. 244, 3038]. Primary negative nuclear Overhauser effects were used to calculate interproton distances on enzyme-bound Mg2+dATP and Mg2+TTP. These distances established that each substrate was bound with an anti-glycosidic torsional angle (chi) of 50 +/- 10 degrees for Mg2+dATP and 40 +/- 10 degrees for Mg2+TTP. The sugar pucker of both substrates was predominantly O1'-endo, with a C5'-C4'-C3'-O3' exocyclic torsional angle (delta) of 95 +/- 10 degrees for Mg2+dATP and 100 +/- 10 degrees for Mg2+TTP. The consistency of these conformations with those previously proposed, on the basis of distances from Mn2+ at the active site [Sloan, D. L., Loeb, L. A., Mildvan, A.S., & Feldman, R.J. (1975) J. Biol. Chem. 250, 8913], indicates a unique conformation for each bound nucleotide. The chi and delta values of the bound substrates are appropriate for nucleotide units of B DNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nuclear Overhauser effect studies of the conformations of MgATP bound to the active and secondary sites of muscle pyruvate kinase

MgATP binds both at the active site (site 1) and at a secondary site (site 2) on each monomer of muscle pyruvate kinase as previously found by binding studies and by X-ray analysis. Interproton distances on MgATP bound at each site have been measured by the time-dependent nuclear Overhauser effect in the absence and presence of phosphoenolpyruvate (P-enolpyruvate), which blocks ATP binding at site 1. Interproton distances at site 2 are consistent with a single conformation of bound ATP with a high antiglycosidic torsional angle (chi = 68 +/- 10 degrees) and a C3'-endo ribose pucker (delta = 90 +/- 10 degrees). Interproton distances at site 1, determined in the absence of P-enolpyruvate by assuming the averaging of distances at both sites, cannot be fit by a single adenine-ribose conformation but require the contribution of at least three low-energy structures: 62 +/- 10% low anti (chi = 30 degrees), C3'-endo; 20 +/- 8% high anti (chi = 55 degrees), O1'-endo; and 18 +/- 8% syn (chi = 217 degrees), C2'-endo. Although a different set of ATP conformations might also have fit the interproton distances, the mixture of conformations used also fits previously determined distances from Mn2+ to the protons of ATP bound at site 1 [Sloan, D. L., & Mildvan, A. S. (1976) J. Biol. Chem. 251, 2412] and is similar to the adenine-ribose portion of free Co(NH3)4ATP, which consists of 35% low anti, 51% high anti, and 14% syn [Rosevear, P. R., Bramson, H. N., O'Brian, C., Kaiser, E. T., & Mildvan, A. S. (1983) Biochemistry 22, 3439].(ABSTRACT TRUNCATED AT 250 WORDS)     

Nuclear overhauser effect studies on the conformation of magnesium adenosine 5'-triphosphate bound to rabbit muscle creatine kinase

Nuclear Overhauser effects were used to determine interproton distances on MgATP bound to rabbit muscle creatine kinase. The internuclear distances were used in a distance geometry program that objectively determines both the conformation of the bound MgATP and its uniqueness. Two classes of structures were found that satisfied the measured interproton distances. Both classes had the same anti glycosidic torsional angle (chi = 78 +/- 10 degrees) but differed in their ribose ring puckers (O1'-endo or C4'-exo). The uniqueness of the glycosidic torsional angle is consistent with the preference of creatine kinase for adenine nucleotides. One of these conformations of MgATP bound to creatine kinase is indistinguishable from the conformation found for Co(NH3)4ATP bound to the catalytic subunit of protein kinase, which also has a high specificity for adenine nucleotides [chi = 78 +/- 10 degrees, O1'-endo; Rosevear, P.R., Bramson, H.N., O'Brian, C., Kaiser, E.T., & Mildvan, A.S. (1983) Biochemistry 22, 3439]. Distance geometry calculations also suggest that upper limit distances, when low enough (less than or equal to 3.4 A), can be used instead of measured distances to define, within experimental error, the glycosidic torsional angle of bound nucleotides. However, this approach does not permit an evaluation of the ribose ring pucker.     

NMR studies of the AMP-binding site and mechanism of adenylate kinase

NMR has previously been used to determine the conformation of enzyme-bound MgATP and to locate the MgATP-binding site on adenylate kinase [Fry, D. C., Kuby, S. A., & Mildvan, A. S. (1985) Biochemistry 24, 4680-4694]. To determine the conformation and location of the other substrate, AMP, distances have been measured from Cr3+AMPPCP, a linear competitive inhibitor with respect to MgATP, to six protons and to the phosphorus atom of AMP on adenylate kinase, with the paramagnetic probe-T1 method. Time-dependent nuclear Overhauser effects (NOEs) have been used to measure five interproton distances on enzyme-bound AMP. These distances were used to determine the conformation of bound AMP in addition to its position with respect to metal-ATP. Enzyme-bound AMP exhibits a high anti-glycosyl torsional angle (chi = 110 +/- 10 degrees), a 3'-endo,2'-exo ribose pucker (delta = 105 +/- 10 degrees), and gauche-trans orientations about the C4'-C5' bond (gamma = 180 +/- 10 degrees) and the C5'-O5' bond (beta = 170 +/- 20 degrees). The distance from Cr3+ to the phosphorus of AMP is 5.9 +/- 0.3 A, indicating a reaction coordinate distance of approximately 3 A, which is consistent with an associative SN2 mechanism for the phosphoryl transfer. Ten intermolecular NOEs, from protons of the enzyme to those of AMP, were detected, indicating the proximity of at least three hydrophobic amino acids to bound AMP. These constraints, together with the conformation of AMP and the intersubstrate distances, were used to position AMP into the X-ray structure of adenylate kinase. The AMP binding site is found to be near (less than or equal to 4 A from) Leu-116, Arg-171, Val-173, Val-182, and Leu-190; all of these residues have been found to be invariant in muscle-type rabbit, calf, human, porcine [Kuby, S. A., Palmieri, R. H., Frischat, A., Fischer, A. H., Wu, L. H., Maland, L., & Manship, M. (1984) Biochemistry 23, 2393-2399], and chicken adenylate kinase [Kishi, F., Maruyama, M., Tanizawa, Y., & Nakazawa, A. (1986) J. Biol. Chem. 261, 2942-2945].     

Conformation of an enzyme-bound substrate of staphylococcal nuclease as determined by NMR.

The dinucleoside phosphodiester dTdA is a slow substrate of staphylococcal nuclease (kcat = 3.8 X 10(-3) s-1) that forms binary E-S and ternary E-M-S complexes with Ca2+, Mn2+, Co2+, and La3+. The enzyme enhances the paramagnetic effects of Co2+ on 1/T1 and 1/T2 of the phosphorus and on 1/T1 of six proton resonances of dTdA, and these effects are abolished by binding of the competitive inhibitor 3',5'-pdTp. From paramagnetic effects of Co2+ on 1/T2 of phosphorus, koff of dTdA from the ternary E-Co(2+)-dTdA complex is greater than or equal to 4.8 X 10(4) s-1 and kon greater than or equal to 1.4 X 10(6) M-1 s-1, indicating the 1/T1 values to be in fast exchange. From paramagnetic effects of enzyme-bound Co2+ on 1/T1 of phosphorus and protons, with use of a correlation time of 1.6 ps on the basis of 1/T1 values at 250 and 600 MHz, 7 metal-nucleus distances and 9 lower-limit metal-nucleus distances are calculated. The long Co2+ to 31P distance of 4.1 +/- 0.9 A, which is intermediate between that expected for direct phosphoryl coordination (3.31 +/- 0.02 A) and a second sphere complex with an intervening water ligand (4.75 +/- 0.02 A), suggests either a distorted inner sphere complex or the rapid averaging of 18% inner sphere and 82% second sphere complexes and may explain the reduced catalytic activity with small dinucleotide substrates. Seventeen interproton distances and 108 lower limit interproton distances in dTdA in the ternary E-La(3+)-dTdA complex were determined by NOESY spectra at 50-, 100-, and 200-ms mixing times. While metal-substrate and interproton distances alone did not yield a unique structure, the combination of both sets of distances yielded a very narrow range of conformations for enzyme-bound dTdA, which was highly extended, with no base stacking, with high-anti glycosidic torsional angles for dT (64 degrees less than or equal to chi less than or equal to 73 degrees) and dA (66 degrees less than or equal to chi less than or equal to 68 degrees) and predominantly C-2'-endo sugar puckers for both nucleosides. Although the individual nucleosides are like those of B-DNA, their unstacked conformation, which is inappropriate for base pairing, as well as the conformational angles alpha and gamma of dA and zeta of dT, rule out B-DNA.(ABSTRACT TRUNCATED AT 400 WORDS)     

Assays for the fidelity of DNA polymerases in cell-free extracts of Escherichia coli are complicated by contaminating nucleoside triphosphatases.

In the presence of DNA and a divalent cation, an enzyme activity in cell-free extracts of Escherichia coli readily hydrolyses dATP to dADP. dGTP is degraded to a smaller extent, dCTP and dTTP being hardly affected. The artificial template primers poly(dC) . oligo(dG) and poly(dT) . oligo(dA) are also effective cofactors for this triphosphatase activity. As a consequence, assays measuring the misincorporation, by cell-free extracts, of dATP and dGTP into these defined templates are difficult to interpret, since the triphosphate substrate is being rapidly degraded during the polymerase reaction. A partial characterization of the dATPase activity was performed, demonstrating that the optimal conditions for its activity resemble those commonly used for assaying polymerase activity. Thus in crude extracts both polymerase and dATPase compete for the same substrate. The inclusion of an ATP-generating system in the reaction mixture maintains the levels of deoxynucleoside triphosphates and changes the kinetics of misincorporation of dAMP into poly(dC) . oligo(dG). No reproducible difference in such misincorporation has been found between lysates prepared from tif-1 cells grown at either permissive or restrictive temperature.     

NMR studies of the MgATP binding site of adenylate kinase and of a 45-residue peptide fragment of the enzyme

Proton NMR was used to study the interaction of beta,gamma-bidentate Cr3+ATP and MgATP with rabbit muscle adenylate kinase, which has 194 amino acids, and with a synthetic peptide consisting of residues 1-45 of the enzyme, which has previously been shown to bind MgepsilonATP [Hamada, M., Palmieri, R. H., Russell, G. A., & Kuby, S. A. (1979) Arch. Biochem. Biophys. 195, 155-177]. The peptide is globular and binds Cr3+ATP competitively with MgATP with a dissociation constant, KD(Cr3+ATP) = 35 microM, comparable to that of the complete enzyme [KI(Cr3+ATP) = 12 microM]. Time-dependent nuclear Overhauser effects (NOE's) were used to measure interproton distances on enzyme- and peptide-bound MgATP. The correlation time was measured directly for peptide-bound MgATP by studying the frequency dependence of the NOE's at 250 and 500 MHz. The H2' to H1' distance so obtained (3.07 A) was within the range established by X-ray and model-building studies of nucleotides (2.9 +/- 0.2 A). Interproton distances yielded conformations of enzyme- and peptide-bound MgATP with indistinguishable anti-glycosyl torsional angles (chi = 63 +/- 12 degrees) and 3'-endo/O1'-endo ribose puckers (sigma = 96 +/- 12 degrees). Enzyme- and peptide-bound MgATP molecules exhibited different C4'-C5' torsional angles (gamma) of 170 degrees and 50 degrees, respectively. Ten intermolecular NOE's from protons of the enzyme and four such NOE's from protons of the peptide to protons of bound MgATP were detected, which indicated proximity of the adenine ribose moiety to the same residues on both the enzyme and the peptide. Paramagnetic effects of beta,gamma-bidentate Cr3+ATP on the longitudinal relaxation rates of protons of the peptide provided a set of distances to the side chains of five residues, which allowed the location of the bound Cr3+ atom to be uniquely defined. Distances from enzyme-bound Cr3+ATP to the side chains of three residues of the protein agreed with those measured for the peptide. The mutual consistency of interproton and Cr3+ to proton distances obtained in metal-ATP complexes of both the enzyme and the peptide suggests that the conformation of the peptide is very similar to that of residues 1-45 of the enzyme. When this was assumed to be the case and when molecular models and a computer graphics system were used, MgATP could be fit into the X-ray structure of adenylate kinase in a unique manner such that all of the distances determined by NMR were accommodated.(ABSTRACT TRUNCATED AT 400 WORDS)     

Nuclear Overhauser effect studies on the conformations of Mg(alpha,beta-methylene)ATP bound to Escherichia coli methionyl-tRNA synthetase

Internuclear distances obtained from nuclear Overhauser effects were used in combination with a distance geometry algorithm to determine the conformation of Mg(alpha,beta-methylene)ATP bound to the Escherichia coli truncated methionyl-tRNA synthetase (delta MTS) both in the absence and presence of cognate and noncognate amino acids. Mg(alpha,beta-methylene)ATP, a nonhydrolyzable analog of ATP, was used to prevent hydrolysis of the nucleotide in the presence of either cognate or noncognate amino acids. Kinetic analysis showed that Mg(alpha,beta-methylene)ATP was a linear competitive inhibitor with respect to ATP in the ATP-pyrophosphate exchange reaction with a Ki = 1.2 mM. The pattern of internuclear Overhauser effects on Mg(alpha,beta-methylene)ATP bound to delta MTS was qualitatively consistent only with an anti glycosidic torsional angle, suggesting that the adenosine portion of the nucleotide is uniquely oriented in the binary enzyme-nucleotide complex. Nearly identical patterns of nuclear Overhauser effects were also observed in ternary complexes containing either cognate L-methionine or noncognate L-homocysteine amino acids. Distance geometry calculations permitted the range and conformational space of the allowed adenine-ribose glycosidic torsional angles in each of the complexes to be better defined and compared. Average adenine-ribose glycosidic torsional angles for enzyme-bound Mg(alpha,beta-methylene)ATP of -106 +/- 9 degrees, -99 +/- 11 degrees, and -97 +/- 11 degrees were determined for the delta MTS.Mg(alpha,beta-methylene)ATP, delta MTS.Mg(alpha,beta-methylene)ATP.L-methionine, and delta MTS.Mg(alpha,beta-methylene)ATP.L-homocysteine complexes, respectively. Comparison of the three enzyme-bound conformations showed that a single nucleotide structure having an adenine-ribose glycosidic torsional angle of -98 degrees with a 3'-endo to O4'-exo ribose sugar pucker was, within error, consistent with the experimental internuclear distances obtained in all three complexes. The nearly identical anti glycosidic torsional angles observed in all three complexes demonstrates that the conformation of the adenosine moiety of the enzyme-bound nucleotide is not sensitive to the presence or the nature of the amino acid bound at the aminoacyladenylate site. Therefore, conformational changes known to occur in the methionyl-tRNA synthetase upon ligand binding appear not to alter the bound conformation of the nucleotide. Information on the conformation and arrangement of substrates bound at the aminoacyladenylate site of delta MTS is necessary for understanding the molecular mechanisms involved in amino acid activation and discrimination.     

Secondary structure of the hybrid poly(rA).poly(dT) in solution. Studies involving NOE at 500 MHz and stereochemical modelling within the constraints of NOE data

One-dimensional nuclear Overhauser effect (NOE) in nuclear magnetic resonance spectroscopy along with stereochemically sound model building was employed to derive the structure of the hybrid poly(rA).poly(dT) in solution. Extremely strong NOE was observed at AH2' when AH8 was presaturated; strong NOEs were observed at TH2'TH2' when TH6 was presaturated; in addition the observed NOEs at TH2' and TH2' were nearly equal when TH6 was presaturated. There was no NOE transfer to AH3' from AH8 ruling out the possibility of (C-3'-endo, low anti chi approximately equal to 200 degrees to 220 degrees) conformation for the A residues. The observed NOE data suggest that the nucleotidyl units in both rA and dT strands have equivalent conformations: C-2'-endo/C-1'-exo, anti chi approximately equal to 240 degrees to 260 degrees. Such a nucleotide geometry for rA/dT is consistent with a right-handed B-DNA model for poly(rA).poly(dT) in solution in which the rA and dT strands are conformationally equivalent. Molecular models were generated for poly(rA).poly(dT) in the B-form based upon the geometrical constraints as obtained from the NOE data. Incorporation of (C-2'-endo pucker, chi congruent to 240 degrees to 260 degrees) into the classical B-form resulted in severe close contacts in the rA chain. By introducing base-displacement, tilt and twist along with concomitant changes in the backbone torsion angles, we were able to generate a B-form for the hybrid poly(rA).poly(dT) fully consistent with the observed NOE data. In the derived model the sugar pucker is C-1'-exo, a minor variant of C-2'-endo and the sugar base torsion is 243 degrees, the remaining torsion angles being: epsilon = 198 degrees, xi = 260 degrees, alpha = 286 degrees, beta = 161 degrees and gamma = 72 degrees; this structure is free of any steric compression and indicates that it is not necessary to switch to C-3'-endo pucker for rA residues in order to accommodate the 2'-OH group. The structure that we have proposed for the polynucleotide RNA-DNA hybrid in solution is in complete agreement with that proposed for a hexamer hybrid in solution from NOE data and is inconsistent with the heteronomous model proposed for the fibrous state.     

Purification and characterization of two new high molecular weight forms of DNA polymerase delta

Two high molecular weight DNA polymerases, which we have designated delta I and delta II, have been purified from calf thymus tissue. Using Bio Rex-70, DEAE-Sephadex A-25, and DNA affinity resin chromatography followed by sucrose gradient sedimentation, we purified DNA polymerase delta I 1400-fold to a specific activity of 10 000 nmol of nucleotide incorporated h-1 mg-1, and DNA polymerase delta II was purified 4100-fold to a final specific activity of 30 000 nmol of nucleotide incorporated h-1 mg-1. The native molecular weights of DNA polymerase delta I and DNA polymerase delta II are 240 000 and 290 000, respectively. Both enzymes have similarities to other purified delta-polymerases previously reported in their ability to degrade single-stranded DNA in a 3' to 5' direction, affinity for an AMP-hexane-agarose matrix, high activity on poly(dA) X oligo(dT) template, and relative resistance to the polymerase alpha inhibitors N2-(p-n-butylphenyl)dATP and N2-(p-n-butylphenyl)dGTP. These two forms of DNA polymerase delta also share several common features with alpha-type DNA polymerases. Both calf DNA polymerase delta I and DNA polymerase delta II are similar to calf DNA polymerase alpha in molecular weight, are inhibited by the alpha-polymerase inhibitors N-ethylmaleimide and aphidicolin, contain an active DNA-dependent RNA polymerase or primase activity, display a similar extent of processive DNA synthesis, and are stimulated by millimolar concentrations of ATP. We propose that calf DNA polymerase delta I, which also has a template specificity essentially identical with that of calf DNA polymerase alpha, could be an exonuclease-containing form of a DNA replicative enzyme.     

Cloning,overexpression, and purification of aminoglycoside antibiotic 3-acetyltransferase-IIIb: conformational studies with bound substrates

Aminoglycoside 3-acetyltransferase-IIIb (AAC3), which acetylates N3 amine of aminoglycoside antibiotics, was cloned from P. Aeruginosa and purified from overexpressing E. coli BL21 (DE3) cells. Bound conformations of kanamycin A and ribostamycin, in the active site of the enzyme that modifies the essential N3B of aminoglycoside antibiotics, were determined by NMR spectroscopy. Experimentally determined interproton distances were used in a simulated annealing protocol to determine enzyme-bound conformations of both antibiotics. Two conformations, consistent with the NOE restraints, were determined for ribostamycin. The only difference between the two conformers was the orientation of the A ring with respect to the rest of the molecule. The average glycosidic dihedral angles were Phi(1A) = -22 degrees +/- 3 and Psi(1A) = -42 degrees +/- 1 (conformer 1) and Phi(1A) = -67 degrees +/- 0.7 and Phi(1A) = -59 degrees +/- 0.8 (conformer 2). Three conformers were determined for the enzyme-bound kanamycin A. Two conformers of kanamycin A were matched well with the two conformers of ribostamycin when the A and the B rings of the antibiotics were superimposed. Conformations of kanamycin A and ribostamycin were compared to those of other aminoglycosides that are bound to different enzymes and RNA. The results lend further support to our earlier hypothesis that the A and B rings of aminoglycosides adopt a conformation that is recognized not only by the aminoglycoside-modifying enzymes but also by RNA (Serpersu, E. H., Cox, J. R., Digiammarino, E. L., Mohler, M. L., Akal, A., Ekman, D. R., and Owston, M. (2000) Cell Biochem. Biophys. 33, 309-321). These results may be useful in designing new antibiotics to combat the antibiotic resistance against infectious diseases.     

The role of Arg165 towards base flipping, base stabilization and catalysis in M.HhaI

Arg165 forms part of a previously identified base flipping motif in the bacterial DNA cytosine methyltransferase, M.HhaI. Replacement of Arg165 with Ala has no detectable effect on either DNA or AdoMet affinity, yet causes the base flipping and restacking transitions to be decreased approximately 16 and 190-fold respectively, thus confirming the importance of this motif. However, these kinetic changes cannot account for the mutant's observed 10(5)-fold decreased catalytic rate. The mutant enzyme/cognate DNA cocrystal structure (2.79 A resolution) shows the target cytosine to be positioned approximately 30 degrees into the major groove, which is consistent with a major groove pathway for nucleotide flipping. The pyrimidine-sugar chi angle is rotated to approximately +171 degrees, from a range of -95 degrees to -120 degrees in B DNA, and -77 degrees in the WT M.HhaI complex. Thus, Arg165 is important for maintaining the cytosine positioned for nucleophilic attack by Cys81. The cytosine sugar pucker is in the C2'-endo-C3'-exo (South conformation), in contrast to the previously reported C3'-endo (North conformation) described for the original 2.70 A resolution cocrystal structure of the WT M.HhaI/DNA complex. We determined a high resolution structure of the WT M.HhaI/DNA complex (1.96 A) to better determine the sugar pucker. This new structure is similar to the original, lower resolution WT M.HhaI complex, but shows that the sugar pucker is O4'-endo (East conformation), intermediate between the South and North conformers. In summary, Arg165 plays significant roles in base flipping, cytosine positioning, and catalysis. Furthermore, the previously proposed M.HhaI-mediated changes in sugar pucker may not be an important contributor to the base flipping mechanism. These results provide insights into the base flipping and catalytic mechanisms for bacterial and eukaryotic DNA methyltransferases.     

A modified version of the Cornell et al. force field with improved sugar pucker phases and helical repeat

We have examined some subtle parameter modifications to the Cornell et al. force field, which has proven quite successful in reproducing nucleic acid properties, but whose C2'-endo sugar pucker phase and helical repeat for B DNA appear to be somewhat underestimated. Encouragingly, the addition of a single V2 term involving the atoms C(sp3)-O-(sp3)-C(sp3)-N(sp2), which can be nicely rationalized because of the anomeric effect (lone pairs on oxygen are preferentially oriented relative to the electron withdrawing N), brings the sugar pucker phase of C2'-endo sugars to near perfect agreement with ab initio calculations (W near 162 degrees). Secondly, the use of high level ab initio calculations on entire nucleosides (in contrast to smaller model systems necessitated in 1994-95 by computer limitations) lets one improve the chi torsional potential for nucleic acids. Finally, the O(sp3)-C(sp3)- C(sp3)-O(sp3) V2 torsional potential has been empirically adjusted to reproduce the ab initio calculated relative energy of C2'-endo and C3'-endo nucleosides. These modifications are tested in molecular dynamics simulations of mononucleosides (to assess sugar pucker percentages) and double helices of DNA and RNA (to assess helical and sequence specific structural properties). In both areas, the modified force field leads to improved agreement with experimental data.     

Conformational microheterogeneity in a DNA double helix: structure of restriction endonuclease Bam H1 recognition site

Structural studies using 500 MHz 1H NMR spectroscopy on Bam H1 recognition site d(GGATCC)2 in solution at 19 degrees is reported. The resonances from the sugar ring and base protons have been assigned from the 2D-COSY and NOESY spectra. Analyses of the NOESY cross-peaks between the base protons H8/H6 and sugar protons H2'/H2", H3' reveal that the nucleotide units G2, A3 and C6 adopt (C3'-endo, chi = 200 degrees-220 degrees) conformation while G1, T4 and C5 exhibit (C2'-endo, chi = 240 degrees-260 degrees) conformation. NMR data clearly suggest that the two strands of d(GGATCC)2 are conformationally equivalent and there is a structural two-fold between the two A-T pairs. The above information and the NOESY data are used to generate a structural model of d(GGATCC)2. The important features are: (i) G1-G2 stack, the site of cleavage, shows an alternation in sugar pucker i.e. C2'-endo, C3'-endo as in a B-A junction, (ii) G2-A3 stack adopts a mini A-DNA, both the sugars being C3'-endo, (iii) A3-T4 stack, the site of two-fold, displays an A-B junction with alternation in sugar pucker as C3'-endo, C2'-endo, (iv) T4-C5 stack adopts a mini B-DNA both the sugars being C2'-endo and (v) C5-C6 stack exhibits a B-A junction with C2'-endo, C3'-endo sugar puckers. Thus, our studies demonstrate that conformational microheterogeneity with a structural two fold, is present in the Bam H1 recognition site.     

Molecular structures of two new anti-HIV nucleoside analogs: 9-(2,3-dideoxy-2-fluoro-beta-D-threo-pentofuranosyl)adenine and 9-(2,3-dideoxy-2-fluoro-beta-D-threo-pentofuranosyl)hypoxanthine.

The x-ray crystal structures of two new anti-HIV compounds, 9-(2,3-dideoxy-2-fluoro-beta-D-threo-pentofuranosyl)adenine (2'-F-dd-araA) and 9-(2,3-dideoxy-2-fluoro-beta-D-threo- pentofuranosyl)hypoxanthine (2'-F-dd-aral), have been determined at two temperatures. Both crystals are in the space group P2(1)2(1)2(1), and their structures were solved by direct methods. Least-squares refinement produced final R-factors of 0.027 for the 2'-F-dd-araA structure and of 0.044 for the 2'-F-dd-aral structure, respectively. The latter structure contains a two-fold disordered conformation of the sugar moiety. All three conformers (one for 2'-F-dd-araA and two for 2'-F-dd-aral) adopt an anti chi CN glycosyl torsion angle. The sugar in the 2'-F-dd-araA structure has a C2'-endo pucker conformation, whereas the sugar in the 2'-F-dd-aral structure has a mixture of C2'-endo and C3'-endo pucker conformations. When the sugar adopts the C2'-endo conformation, the torsion angle about the C4'-C5' bond is in a transgauche+ conformation. In contrast, when the sugar adopts the C3'-endo conformation, the torsion angle about the C4'-C5' bond is in a gauche(+)-gauche- conformation. The C2'-F bond distance is 1.406(3) A, similar to that found in other aliphatic C-F bonds. The results suggest that the 2'-fluoro-2',3'-dideoxyarabinosyl nucleosides do not have a strong preference for either C2'-endo or C3'-endo sugar pucker.     

Unusual nucleotide conformations in GNRA and UNCG type tetraloop hairpins: evidence from Raman markers assignments.

High resolution NMR data on UNCG and GNRA tetraloops (where N is any of the four nucleotides and R is a purine) have shown that they contain ribonucleosides with unusual 2'-endo/anti and 3'-endo/syn conformations, in addition to the 3'-endo/anti ones which are regularly encountered in RNA chains. In the current study, Raman spectroscopy has been used to probe these nucleoside conformations and follow the order (hairpin) to disorder (random chain) structural transitions in aqueous phase in the 5-80 degreesC temperature range. Spectral evolution of GCAA and GAAA tetraloops, as formed in very short hairpins with only three G.C base pairs in their stems (T m >60 degreesC), are reported and compared with those previously published on UUCG and UACG tetraloops, for which the syn orientation of the terminal guanine as well as the 2'-endo/anti conformation of the third rC residue have been confirmed by means of vibrational marker bands. Raman data obtained as a function of temperature show that the first uracil in the UUCG tetraloop is stacked and the two middle residues (rU and rC) are in the 2'-endo/anti conformation, in agreement with the previously published NMR results. As far as the new data concerning the GNRA type tetraloops are concerned, they lead us to conclude that: (i) in both cases (GCAA and GAAA tetraloops) the adenine bases are stacked; (ii) the second rC residue in the GCAA tetraloop has a 3'-endo/anti conformation; (iii) the sugar pucker associated with the third rA residue in both tetraloops possibly undergoes a 3'-endo/2'-endo interconversion as predicted by NMR results; (iv) the stem adopts a regular A-form structure; (v) all other nucleosides of these two GNRA tetraloops possess the usual 3'-endo/anti conformation.     

Interpretation of DNA vibration modes: I--The guanosine and cytidine residues involved in poly(dG-dC).poly(dG-dC) and d(CG)3.d(CG)3

A normal coordinate analysis has been carried out on guanosine and cytidine residues appearing in oligo and polynucleotides by using a simplified valence force field that allows the vibrational spectra of 5'-dGMP and 2'-deoxycytidine molecules to be reproduced. The role of both C2'-endo and C3'-endo conformations on sugar pucker, as well as that of glycosidic torsion angle (X), on several characteristic vibration modes of these residues have been studied. The present calculations based on a non-redundant set of internal coordinates preserving the harmonic approximation of the potential field, allows us to explain quite satisfactorily the modifications of the vibrational spectra in the 1550-1250 cm-1 and 785-500 cm-1 regions, when the right----left-handed conformational transition occurs.