Biomedical subjects
Publications and source records attributed to A Galeone.
The preparation of a solid support useful for the synthesis of oligonucleotides with a 3'-3' inversion of polarity, via a linker containing a chelating molecule, namely 2,2'-bipyridine, is described.
2'-Deoxy-8-(propyn-1-yl)adenosine has been incorporated in synthetic oligodeoxyribonucleotides and its influence on thermal stability of duplex and quadruplex structures investigated by UV, CD and 1H NMR. The obtained results seem to indicate that the presence of the modified base negatively affects the stability of double stranded DNA whereas remarkably increases the stability of parallel quadruplex structures.
A new polymeric prodrug was prepared coupling 5-iodo-2'-deoxyuridine (IDU) to poly(d,l-lactic acid) (PLA) via a succinic acid spacer. The PLA-IDU conjugate was characterised by thermal analysis, IR and 1H and 13C NMR spectroscopy. The IDU content (0.024 mequiv.g-1 of PLA) was consistent with the carboxylic acid endgroup present in the polymer sample (0.025 mequiv.g-1 of polymer). The PLA-IDU conjugate was susceptible to degradation in biological environments containing esterase, whereas IDU was not detected by chemical hydrolysis in pH 7.4 phosphate buffer. The conjugate should be used to prepare injectable microspheres and nanospheres containing IDU chemically coupled to the polymer carrier.
The TATA binding protein (TBP), which plays a central role in gene regulation as an essential component of all three nuclear transcription systems, sharply kinks the TATA box at two sites and severely contorts the intervening DNA segment. DNA constructs with precisely localized flexure have been used to investigate the special repertoire of mechanisms and properties that arise from TBP interacting with the TATA box. DNA flexure precisely localized to the sites of TBP-mediated DNA kinking increases the affinity of TBP more than 100-fold; unexpectedly, this increase in affinity is achieved almost exclusively by increasing the stability of the TBP-DNA complex rather than the rate of its formation. In vitro transcription with RNA polymerase III provides a first demonstration that the orientation of TBP on the TATA box is governed by DNA deformability, its C-proximal repeat contacting the more flexible end of the TATA box. Exceptionally stable TBP-DNA complexes reach their orientational equilibrium very slowly; in these circumstances, assembly of stable ("committed") transcription initiation complexes can freeze far-from-equilibrium orientations of TBP on the TATA box, causing transcription polarity to be determined by a kinetic trapping mechanism.
The DNA Holliday junction is a central intermediate in genetic recombination. We have designed and synthesized a DNA oligomer, J1a, as a model compound for the Holliday junction suitable to be studied by NMR spectroscopy and future molecular modelling. The design was based on a 46-base oligomer, J4, previously studied by Pikkemaat, J. A., van den Elst, H., van Boom, J. H. & Altona, C. [Biochemistry 33, 14896-14907 (1994)], including the propensity to undergo a self-folding process to give a four-way junction in which three of the four arms are capped with a hairpin loop. J1a, however, is considerably shortened by eight bases and thus contains only 38 residues which significantly facilitates the proton resonance assignments. The base sequence at the branch point is identical to that in J4. 1H-NMR data clearly point to the presence of three hairpin loops in J1a and show that the double-helical arms adopt the B-DNA form. Quasicontinuous pairwise stacking between helical arms to give a single preferred stacked X-conformation is evident. The extent of folding into this stacked conformation is strongly dependent upon the magnesium concentration. Full Watson-Crick base pairing at the branch point is completely preserved. The A/D-stacking preference of the small junction is the same as that exhibited by J4.
The DNA-bending protein TF1 is the Bacillus subtilis bacteriophage SPO1-encoded homolog of the bacterial HU proteins and the Escherichia coli integration host factor. We recently proposed that TF1, which binds with high affinity (Kd was approximately 3 nM) to preferred sites within the hydroxymethyluracil (hmU)-containing phage genome, identifies its binding sites based on sequence-dependent DNA flexibility. Here, we show that two hmU-A base pair steps coinciding with two previously proposed sites of DNA distortion are critical for complex formation. The affinity of TF1 is reduced 10-fold when both of these hmU-A base pair steps are replaced with A-hmU, G-C, or C-G steps; only modest changes in affinity result when substitutions are made at other base pairs of the TF1 binding site. Replacement of all hmU residues with thymine decreases the affinity of TF1 greatly; remarkably, the high affinity is restored when the two hmU-A base pair steps corresponding to previously suggested sites of distortion are reintroduced into otherwise T-containing DNA. T-DNA constructs with 3-base bulges spaced apart by 9 base pairs of duplex also generate nM affinity of TF1. We suggest that twin hmU-A base pair steps located at the proposed sites of distortion are key to target site selection by TF1 and that recognition is based largely, if not entirely, on sequence-dependent DNA flexibility.
The pyrimidine base 5-(hydroxymethyl)-2'-deoxyuridine (HmU) is a common nucleotide in SPO1 phage DNA. Numerous transcriptional proteins bind HmU-containing DNA preferentially implicating a regulatory function of HmU. We have investigated the conformation and dynamics of d-(5'-CHmUCHmUACACGHmUGHmUAGAG-OH-3')2 (HmU-DNA). This oligonucleotide mimics the consensus sequence of Transcription Factor 1 (TF1). The HmU-DNA was compared to the thymine-containing oligonucleotide. NOESY and DQF COSY spectroscopy provided resonance assignments of nonexchangeable and exchangeable protons, intranucleotide, internucleotide and intrastrand proton-proton distances, and dihedral angle constraints. Methylene protons of the hydroxymethyl group are nonequivalent protons and the hydroxymethyl group is not freely rotating. The hydroxymethyl group adopts a specific orientation with the OH group oriented on the 3' side of the plane of the base. Analysis of imino proton resonances and NOEs indicates additional end base pair fraying and a temperature-induced transition to a conformation in which the internal HmU-A base pairs are disrupted or have reduced lifetimes. Orientation of the hydroxymethyl group indicates the presence of internucleotide intrastrand hydrogen bonding between the HmU12C5 hydroxyl group and A13. All sugars in both DNAs show a C2'endo conformation (typical of B-DNA).
Certain DNA-binding proteins function as architectural elements by bending DNA. We have studied the binding of three such proteins, the prokaryotic HU and integration host factor (IHF) and the eukaryotic HMG1, to DNA in which flexibility is enhanced by tandem mismatches and by substituting 5-hydroxymethyluracil (hmU) for thymine (T). IHF and HU have higher affinity for DNA with two 4-nt loops than for perfect duplex DNA with a sequence that corresponds to a binding site for the phage-encoded homolog, TF1. HU has a high affinity for DNA with 4-nt loops separated by 9 bp (Kd = 3.5 nM), with suboptimal binding for other loop separations. IHF-binding is optimal when 4-nt loops are 8 to 9 bp apart; optimal complex formation with DNA representing the specific IHF-binding site H' requires that loops do not disrupt the consensus sequence and that one 4-nt loop borders the dyad axis-proximal block of consensus sequence (Kd = 0.3 nM, approximately tenfold lower than for H' perfect duplex DNA). HMG1 also binds preferentially to DNA with loops. All three proteins bind more tightly to DNA in which thymine is replaced with hmU. IHF has a tenfold higher affinity for hmU-DNA without a consensus IHF site (Kd = 7.6 nM) than for the corresponding T-DNA but does exhibit site-selectivity in hmU-DNA; Kd = 0.6 nM for the hmU-containing version of H'. Tighter binding to hmU-DNA is consistent with greater flexibility, and the distinct influence of loop position on complex formation suggests that sequence-dependent variations in flexibility of duplex DNA play a significant role in target-site selection by these DNA-bending proteins.
TF1 is a member of the family of type II DNA-binding proteins, which also includes the bacterial HU proteins and the Escherichia coli integration host factor (IHF). Distinctive to TF1, which is encoded by the Bacillus subtilis bacteriophage SPO1, is its preferential binding to DNA in which thymine is replaced by 5-hydroxymethyluracil (hmU), as it is in the phage genome. TF1 binds to preferred sites within the phage genome and generates pronounced DNA bending. The extent to which DNA flexibility contributes to the sequence-specific binding of TF1, and the connection between hmU preference and DNA flexibility has been examined. Model flexible sites, consisting of consecutive mismatches, increase the affinity of thymine-containing DNA for TF1. In particular, tandem mismatches separated by nine base-pairs generate an increase, by orders of magnitude, in the affinity of TF1 for T-containing DNA with the sequence of a preferred TF1 binding site, and fully match the affinity of TF1 for this cognate site in hmU-containing DNA (Kd approximately 3 nM). Other placements of loops generate suboptimal binding. This is consistent with a significant contribution of site-specific DNA flexibility to complex formation. Analysis of complexes with hmU-DNA of decreasing length shows that a major part of the binding affinity is generated within a central 19 bp segment (delta G0 = 41.7 kJ mol-1) with more-distal DNA contributing modestly to the affinity (delta delta G = -0.42 kJ mol-1 bp-1 on increasing duplex length to 37 bp). However, a previously characterised thermostable and more tightly binding mutant TF1, TF1(E15G/T32I), derives most of its extra affinity from interaction with flanking DNA. We propose that inherent but sequence-dependent deformability of hmU-containing DNA underlies the preferential binding of TF1 and that TF1-induced DNA bendings is a result of distortions at two distinct sites separated by 9 bp of duplex DNA.
In the course of a study aimed at the synthesis of pyrrole amidine carboxamide DNA-binding agents as novel pharmacological agents, a series of carbamoyl analogues of distamycin, containing an increasing number of pyrrole units, have been obtained by total synthesis. The interaction of the tetrapyrrole carbamoyl 4 with the dodecamer d(CGCAAATTTGCG)2 in comparison with that of the corresponding formylamino analogue 3 has been examined by high-resolution 1H-NMR and molecular modeling. Either ligand binds to DNA in one-drug and symmetric two-drug modes at low drug:DNA ratios, while at high ratios only the two-drug complex was observed. In this article, the structure of 2:1 drugs DNA complexes has been studied by NMR and molecular modeling, which indicate that the two analogues bind the DNA in a similar fashion, in the minor groove of the 5'-AATTT region. In both complexes the two drugs are symmetrically placed along the complementary strands of DNA with the pyrrole ring of one molecule in close contact with those of the other one. Although another region of five consecutive A-T base pairs is available, no evidence of sliding of drug molecules between different binding sites, as in the case of the 2:1 complex of distamycin with the same dodecamer, is observed, thus indicating that increasing the number of N-methylpyrrolecarboxamide units from three to four cases a lengthening of the recognition sequence.
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.
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)
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.
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TF1, a homodimeric DNA-binding and -bending protein with a preference for hydroxymethyluracil-containing DNA is the Bacillus subtilis-encoded homolog of the bacterial HU proteins and of the E. coli integration host factor. A temperature-sensitive mutation at amino acid 25 of TF1 (L25-->A) and two intragenic second site revertants at amino acids 15 (E15-->G) and 32 (L32-->I) were previously identified and their effects on virus development were examined. The DNA-binding properties of these proteins and the thermal stability of their secondary structures have now been analyzed. Amino acids 15 and 32 are far removed from the putative DNA-binding domains of TF1 but changes there exert striking effects on DNA affinity that correlate with effects on structure. The double mutant protein TF1-G15I32 binds to a preferred site in hydroxymethyluracil-containing DNA 40 times more tightly, denatures at higher temperature (delta tm = 21 degrees C), and also exchanges subunits much more slowly than does the wild-type protein. The L25-->A mutation makes TF1 secondary structure and DNA-binding highly salt concentration-dependent. The E15-->G mutation partly suppresses this effect: secondary structure of TF1-A25G15 is restored at 21 degrees C by 1 M NaCl or, at low NaCl concentration, by binding to DNA.
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.
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The great progress that has been made in medical knowledge in the last fifty years has led to the birth of various new medical specialities and, consequently, to the institution within hospitals of specialised departments and services. A little at a time, General Medicine has lost responsibility for almost all the main sectors of medical pathology, with the result that there has been a serious deterioration in the importance and prestige of the discipline. Today there is a tendency to consider General Medicine departments as a direct hospital projection of everyday medical practice and hospital medicine is required to fill in the serious gaps in extra-hospital welfare and medical structures. The image and functions of General Medicine within hospitals need reassessing. Major hospitals, which already house the main specialist wards, should be given specialist Internal Medicine departments too. A limitation should also be set on the number of unjustified admittances (which mainly go to General Medicine wards) by means of efficient registration services able to filter patients. General Medicine has a number of important tasks outside the diagnosis and treatment of hospitalised patients. It has an important didactic function geared to the practical medical training of students in their final years, of newly graduated doctors, of those specialising in internal medicine, those specialising in clinical and scintific research, and in the updating of general practitioners outside the hospitals.