Review of methods in "breast augmentation: a risk factor for breast cancer?".
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Bias↗
Biomedical subjects
J H van de Sande
Publications and source records attributed to J H van de Sande.
Interaction of recA protein with left-handed Z-DNA.
The ability of recA protein to interact with a Z-DNA polymer, Br-poly(dG-dC), or M13 bacteriophage single-stranded DNA was investigated. RecA protein binds more avidly to Z-DNA than to single-stranded DNA in the absence of a nucleotide cofactor. This binding pattern changes in the presence of adenosine 5'-(gamma-thio)triphosphate (ATP[S]), however, such that the binding to Z-DNA decreases while binding to single-stranded DNA increases roughly 2-fold. When present together, the two forms of DNA compete with each other in the presence of ATP[S]. Experiments involving recA protein binding to recombinant plasmids showed neither a preferential binding of recA protein to the plasmid containing Z-DNA nor a similar effect of ATP[S] to that observed with the Z-DNA polymer. In contrast, maximal binding was obtained with a plasmid (linear or supercoiled) containing a polypurine.polypyrimidine insert, thus suggesting that recA protein displays sequence preferences in its interaction with DNA. The results of the present study provide no evidence that recA protein specifically interacts with or stabilizes the Z-DNA insert of a recombinant plasmid in the left-handed conformation.
Adenosine Triphosphate↗
Specificities and kinetics of uracil excision from uracil-containing DNA oligomers by Escherichia coli uracil DNA glycosylase.
Uracil DNA glycosylase excises uracil residues from DNA that can arise as a result of deamination of cytosine or incorporation of dUMP residues by DNA polymerase. We have carried out a detailed study to define the specificities and the kinetic parameters for its substrates by using a number of synthetic oligodeoxyribonucleotides of varying lengths and containing uracil residue(s) in various locations. The results show that the Escherichia coli enzyme can remove a 5'-terminal U from an oligomer only if the 5'-end is phosphorylated. The enzyme does not remove U residues from a 3'-terminal position, but U residues can be excised from oligonucleotides with either pd(UN)p or pd(UNN) 3'-termini. The oligomer d(UUUUT) can have the second or third U residues from the 5'-end excised even when the neighboring site is an abasic site (3' or 5', respectively). On the basis of these findings, pd(UN)p was anticipated to be the smallest size substrate. Results show detectable amounts of U release from the substrate pd(UT)p; however, significantly higher amounts of U release were observed from pd(UT-sugar) or pd(UTT). Determinations of the Km and Vmax values show that the different rates of U excision from oligomers of different sizes (trimeric to pentameric) but containing U in the same position are largely due to the differences in the Km values, whereas the different rates of U excision from the substrates of the same size but containing U in different positions are largely due to different Vmax values.
DNA Glycosylases↗
Identification of novel single-stranded d(TC)n binding proteins in several mammalian species.
A group of single-stranded d(TC)n specific binding proteins has been detected in the nuclear extracts of several mammalian species that included mouse, human, African green monkey, chimpanzee, and Chinese muntjac. Southwestern analysis of 500 mM KCI nuclear extracts has shown that these proteins cluster in a similar size range, 55.5 to 57 kD. An additional 54 kD band was present for the three primate species examined. The single-stranded d(TC)n binding activity was confirmed with bandshift assay. Specific double-stranded binding activity for duplex d(TC)n.d(GA)n or single-stranded d(GA)n was not detected. The conservation of size distribution and d(TC)n-binding activity across the species examined indicates that this class of single-stranded binding proteins may have an important biological function in vivo.
Animals↗
Length-dependent formation of parallel-stranded DNA in alternating AT segments.
Parallel-stranded DNA can be formed from alternating AT segments and is not restricted exclusively to homooligomeric AT sequences. DNA oligonucleotides 3'-d(AT)nxC4(AT)n-3' (where x indicates the location of the 5'-5' phosphodiester linkage) form parallel-stranded hairpin structures at micromolar strand concentration for n = 4 or 5 but not for n = 6, 7. The spectral properties of the parallel-stranded structures are similar to those of the hairpin structures containing homooligomeric AT stems. However, parallel-stranded structures formed in alternating AT segments are significantly less stable than either their corresponding antiparallel control or the homooligomeric parallel AT hairpins as evidenced by their lower helix-coil transition enthalpy, melting temperature, and stability constant. This results in a remarkable polymorphism which is most pronounced for 3'-d(AT)5xC4(AT)5-3'. This oligonucleotide can exist as a parallel-stranded hairpin, coil, or concatameric antiparallel structure(s), depending on temperature and strand concentration. These results suggest simple guidelines for the design of parallel-stranded DNA. In addition, we present a model for the assessment of the stability of parallel-stranded duplex structures formed from AT base pairs based on their sequence.
Base Sequence↗
Solution conformation of purine-pyrimidine DNA octamers using nuclear magnetic resonance, restrained molecular dynamics and NOE-based refinement.
The solution structures of two alternating purine-pyrimidine octamers, [d(G-T-A-C-G-T-A-C)]2 and the reverse sequence [d(C-A-T-G-C-A-T-G)]2, are investigated by using nuclear magnetic resonance spectroscopy and restrained molecular dynamics calculations. Chemical shift assignments are obtained for non-exchangeable protons by a combination of two-dimensional correlation and nuclear Overhauser enhancement (NOE) spectroscopy experiments. Distances between protons are estimated by extrapolating distances derived from time-dependent NOE measurements to zero mixing time. Approximate dihedral angles are determined within the deoxyribose ring from coupling constants observed in one and two-dimensional spectra. Sets of distance and dihedral determinations for each of the duplexes form the bases for structure determination. Molecular dynamics is then used to generate structures that satisfy the experimental restraints incorporated as effective potentials into the total energy. Separate runs start from classical A and B-form DNA and converge to essentially identical structures. To circumvent the problems of spin diffusion and differential motion associated with distance measurements within molecules, models are improved by NOE-based refinement in which observed NOE intensities are compared to those calculated using a full matrix analysis procedure. The refined structures generally have the global features of B-type DNA. Some, but not all, variations in dihedral angles and in the spatial relationships of adjacent base-pairs are observed to be in synchrony with the alternating purine-pyrimidine sequence.
Base Sequence↗
Perturbation of DNA hairpins containing the EcoRI recognition site by hairpin loops of varying size and composition: physical (NMR and UV) and enzymatic (EcoRI) studies.
We have investigated loop-induced structural perturbation of the stem structure in hairpins d(GAATTCXnGAATTC) (X = A, T and n = 3, 4, 5 and 6) that contain an EcoRI restriction site in close proximity to the hairpin loop. Oligonucleotides containing either a T3 or a A3 loop were not hydrolyzed by the restriction enzyme and also showed only weak binding to EcoRI in the absence of the cofactor Mg2+. In contrast, hairpins with larger loops are hydrolyzed by the enzyme at the scission site next to the loop although the substrate with a A4 loop is significantly more resistant than the oligonucleotide containing a T4 loop. The hairpin structures with 3 loop residues were found to be thermally most stable while larger hairpin loops resulted in structures with lower melting temperatures. The T-loop hairpins are thermally more stable than the hairpins containing the same number of A residues in the loop. As judged from proton NMR spectroscopy and the thermodynamic data, the base pair closest to the hairpin loop did form in all cases studied. The hairpin loops did, however, affect the conformation of the stem structure of the hairpins. From 31P and 1H NMR spectroscopy we conclude that the perturbation of the stem structure is stronger for smaller hairpin loops and that the extent of the perturbation is limited to 2-3 base pairs for hairpins with T3 or A4 loops. Our results demonstrate that hairpin loops modulate the conformation of the stem residues close to the loop and that this in turn reduces the substrate activity for DNA sequence specific proteins.
DNA↗
Distribution of CT-rich tracts is conserved in vertebrate chromosomes.
The distribution of d(CT)-rich pyrimidine tracts in the karyotypes of a variety of vertebrates was studied by in situ hybridization. The probe for these studies was a 56bp homopyrimidine/homopurine sequence obtained from a mouse genomic library constructed with DNA prepared from a restriction enzyme digestion of metaphase chromosomes. Single-stranded DNA nuclease digestions and two-dimensional gel analysis of topoisomers of this sequence indicated that it is capable of adopting a triplex conformation in vitro. In situ hybridization with this probe to the karyotypes of ten different vertebrate species revealed a highly conserved chromosomal distribution of d(CT)-rich tracts. These tracts are found throughout the chromosomal arms and in some karyotypes they are clustered, producing a banding pattern. However, at the resolution of the light microscope these tracts appeared to be absent from the centromeric regions of all chromosomes examined except those of chicken. The non-random distribution of these tracts to the chromosomal arm regions implies an organizational or functional role for this repeat class. It is unlikely that the 56 bp sequence type contributed to the formation of the triplex DNA structure previously detected in centromeric domains of mouse.
Animals↗
Are many Z-DNA binding proteins actually phospholipid-binding proteins?
We used a Z-DNA affinity column to isolate a collection of Z-DNA binding proteins from a high salt extract of Escherichia coli. We identified one of the major Z-DNA binding proteins of this fraction, not as a protein involved in gene regulation or genetic recombination, but rather as an outer membrane porin protein. We then showed that several other known phospholipid-binding proteins (bovine lung annexins and human serum lipoproteins) also bind much more tightly to Z-DNA than to B-DNA. In all cases, this Z-DNA binding was strongly blocked by competition with acidic phospholipids, such as cardiolipin. Our results raise the question whether many of the Z-DNA binding proteins previously isolated are actually phospholipid-binding proteins.
Carrier Proteins↗
Interaction of RecA protein with acidic phospholipids inhibits DNA-binding activity of RecA.
The RecA protein of Escherichia coli binds specifically to acidic phospholipids such as cardiolipin and phosphatidylglycerol. This binding appears to be affected by the presence of divalent cations such as Ca2+ and Mg2+. The interaction leads to the inhibition of RecA binding to at least two different conformations of DNA, single-stranded DNA and left-handed Z-DNA, thus suggesting that the phospholipids interact at the DNA-binding site of the RecA protein. Inclusion of a nucleotide cofactor [adenosine 5'-O-(gamma-thiotriphosphate)] in the reactions did not prevent the inhibition of DNA-binding activities of RecA protein by the phospholipids. The interaction of RecA protein with cardiolipin and phosphatidylglycerol, which represent two of the three major phospholipids of the E. coli membrane, may be physiologically important, as it provides a possible mechanism for the RecA-membrane association during the SOS response. These observations raise the possibility that the Z-DNA-binding activity of RecA protein is merely a manifestation of its phospholipid-binding property.
DNA↗
Characterization of a parallel-stranded DNA hairpin.
Recently we have shown that synthetic DNA containing homooligomeric A-T base pairs can form a parallel-stranded intramolecular hairpin structure [van de Sande et al. (1988) Science (Washington, D.C.) 241, 551-557]. In the present study, we have employed NMR and optical spectroscopy to investigate the structure of the parallel-stranded (PS) DNA hairpin 3'-d(T)8C4(A)8-3' and the related antiparallel (APS) hairpin 5'-d(T)8C4(A)8-3'. The parallel orientation of the strands in the PS oligonucleotide is achieved by introducing a 5'-5' phosphodiester linkage in the hairpin loop. Ultraviolet spectroscopic and fluorescence data of drug binding are consistent with the formation of PS and APS structures, respectively, in these two hairpins. Vacuum circular dichroism measurements in combination with theoretical CD calculations indicate that the PS structure forms a right-handed helix. 31P NMR measurements indicate that the conformation of the phosphodiester backbone of the PS structure is not drastically different from that of the APS control. The presence of slowly exchanging imino protons at 14 ppm and the observation of nuclear Overhauser enhancement between imino protons and the AH-2 protons demonstrate that similar base pairing and base stacking between T and A residues occur in both hairpins. However, the small chemical shift dispersion observed in proton NMR spectra of the PS hairpin suggests that the stem of this hairpin is more regular than that of the APS hairpin. On the basis of NOESY measurements, we find that the orientation of the bases is in the anti region and that the sugar puckering is in the 2'-endo range.(ABSTRACT TRUNCATED AT 250 WORDS)
Base Sequence↗
Characterization of the ung1 mutation of Escherichia coli.
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Base Sequence↗
Yolk proteins from nematodes, chickens, and frogs bind strongly and preferentially to left-handed Z-DNA.
Yolk proteins purified from the nematode Caenorhabditis elegans, from the frog Xenopus laevis, and from chicken eggs all have the unexpected property of binding strongly and preferentially to a left-handed Z-DNA probe, brominated poly(dG-dC). We estimate that the nematode proteins bind to Z-DNA with an association constant of at least 10(4) (M-1) and that this association constant is at least 40-50-fold higher than the association constant to B-DNA. Thus, yolk proteins have a higher Z-DNA specificity than most of the Z-DNA binding proteins previously isolated from other sources. Although yolk protein binding to Z-DNA is poorly competed by a wide variety of nucleic acids, the interaction is strongly competed by the phospholipids cardiolipin and phosphatidic acid (500-1000-fold better than by the same mass of B-DNA). We suggest that Z-DNA interacts with the yolk protein phospholipid binding site. In general, our results emphasize the danger of using physical properties to infer biological function. In particular, our results should raise serious questions about the biological relevance of previously isolated Z-DNA binding proteins.
Animals↗
Relative stability of parallel- and antiparallel-stranded duplex DNA.
We have recently shown that DNA containing homopolymeric A-T base pairs can form a parallel-stranded intramolecular duplex [van de Sande et al. (1988) Science (Washington, D.C.) 241, 551-557]. In the present paper, we demonstrate that parallel-stranded DNA can also be formed in unconstrained bimolecular DNA of appropriate sequence homology. Three deoxyoligonucleotides, a 21-mer [dCCCATATATATTTTTTTTCCC], a ps-15-mer [dTATATATAAAAAAAA], and an aps-15-mer [dAAAAAAAATATATAT], have been synthesized. Annealing of 21-mer and aps-15-mer results in the formation of a conventional antiparallel duplex (aps); however, the combination of 21-mer and ps-15-mer forms a duplex in which the two strands are in a parallel orientation (ps). The parallel-stranded structure was established from the following criteria: (i) The parallel-stranded structure shows a 1:1 stoichiometry of the constituent strands. (ii) Gel electrophoretic mobility of the ps and aps duplexes are similar under native conditions. (iii) Spectroscopic properties of the ps duplex are characteristics for a base-paired structure but are different from the aps structure. (iv) Both duplexes undergo a thermally induced helix to coil transition; however, the melting temperature for the ps duplex is 22 degrees C lower. (v) The minor groove binding drug Hoechst 33258 shows a reduced affinity for the ps compared to the aps duplex. (vi) The parallel-stranded duplex is not a substrate for DNA Escherichia coli polymerase I (Klenow fragment) or AMV reverse transcriptase. Parallel-stranded DNA can exist under normal solution conditions, but competition experiments show it to be thermodynamically less favorable than the conventional antiparallel form.
Base Composition↗
Comparison of the B- and Z-form hairpin loop structures formed by d(CG)5T4(CG)5.
The partially self-complementary synthetic DNA oligonucleotide d(CG)5T4(CG)5 has been studied by using 1H and 31P NMR and circular dichroism. Results show that, under low-salt conditions (120 mM NaCl buffer), an intramolecular hairpin loop exists in which the double-helical stem region is B-form and the thymidine loop residues have predominantly southern (C2'-endo) sugar conformations. The thymidine glycosidic torsion angles are intermediate between syn and anti or exist as an equilibrium mixture of residues in the two extremes. NOESY data indicate that the structure of the loop region is very similar to that found for d(CG)2T4(CG)2 [Hare, D. R., & Reid, B. R. (1986) Biochemistry 25, 5341-5350]. Under high-salt conditions (6 M NaClO4 buffer), the dominant form (approximately equal to 85%) is an intramolecular hairpin structure in which the stem region forms a Z-form double helix. As in the B-form, the loop thymidine residues are intermediate between the syn and anti conformations or exist as an equilibrium mixture of the two, but the thymidine sugar conformations differ in that they are biased toward northern (C3'-endo) conformations.
Base Sequence↗
Parallel stranded DNA.
A series of four hairpin deoxyoligonucleotides was synthesized with a four-nucleotide central loop (either C or G) flanked by the complementary sequences d(T)10 and d(A)10. Two of the molecules contain either a 3'-p-3' or 5'-p-5' linkage in the loop, so that the strands in the stem have the same, that is, parallel (ps) polarity. The pair of reference oligonucleotides have normal phosphodiester linkages throughout and antiparallel (aps) stem regions. All the molecules adopt a duplex helical structure in that (i) the electrophoretic mobilities in polyacrylamide gels of the ps and aps oligomers are similar. (ii) The ps hairpins are substrates for T4 polynucleotide kinase, T4 DNA ligase, and Escherichia coli exonuclease III. (iii) Salt-dependent thermal transitions are observed for all hairpins, but the ps molecules denature 10 degrees C lower than the corresponding aps oligomers. (iv) The ultraviolet absorption and circular dichroism spectra are indicative of a base-paired duplex in the stems of the ps hairpins but differ systematically from those of the aps counterparts. (v) The bis-benzimidazole drug Hoechst-33258, which binds in the minor groove of B-DNA, exhibits very little fluorescence in the presence of the ps hairpins but a normal, enhanced emission with the aps oligonucleotides. In contrast, the intercalator ethidium bromide forms a strongly fluorescent complex with all hairpins, the intensity of which is even higher for the ps species. (vi) The pattern of chemical methylation is the same for both the ps and aps hairpins. The combined results are consistent with the prediction from force field analysis of a parallel stranded right-handed helical form of d(A)n.d(T)n with a secondary structure involving reverse Watson-Crick base pairs and a stability not significantly different from that of the B-DNA double helix. Models of the various hairpins optimized with force field calculations are described.
DNA↗
Sequence analysis, expression, and conservation of Escherichia coli uracil DNA glycosylase and its gene (ung).
The complete nucleotide sequence of the Escherichia coli ung gene is described. Transcription initiation and termination sites were determined by S1 nuclease and RNase mapping. The common prokaryotic -35, -10, and the ribosome binding site sequences are represented by TGTTCTGTA, TAAGCTA, and AGGAGAG at their respective locations. A putative hairpin transcription terminator structure is present at the major transcription terminator sites. The open reading frame of the ung gene codes for a protein of 229 amino acids (25,664 daltons). The molecular weight, amino acid composition, and the N-terminal amino acid sequence of the uracil DNA glycosylase purified from E. coli cells match with the open reading frame of the ung gene. The protein sequence analysis shows that the N-terminal methionine is cleaved off in the mature protein. The in vitro transcription coupled translation of the ung gene directs the synthesis of a protein which comigrates with uracil DNA glycosylase. Also, the CNBr cleavage of the protein synthesized in vitro confirms the positions of the methionines deduced from the DNA sequence. The levels of ung gene expression remain constant up to the early stationary phase, but decline in the late stationary phase of the E. coli culture. The E. coli gene showed a strong sequence homology to Shigella, a weak sequence homology to Salmonella and Citrobacter, and a very weak sequence homology to Proteus genes. No sequence homologies were seen for Pseudomonas, Clostridium, Micrococcus, and several eukaryotic genomes.
Amino Acid Sequence↗
Inosine incorporation in GC rich RNA probes increases hybridization sequence specificity.
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Cytosine↗