Gerald A. Thomas

Laser Raman Microscopy of Chromosomes in Living Eukaryotic Cells: DNA Polymorphism In Vivo

In order to answer the question of what the structure of DNA is in the nucleus of a living cell where its long length must be subjected to repeated folds in order to pack it into such a small space, a laser Raman microscope was designed and built to take the Raman spectra of DNA in the packaged state in which it occurs in nature. Spectra were taken of the DNA in salmon sperm, squid sperm and the nucleus of the salivary gland of Drosophila melano-gaster. These measurements show that the majority of the deoxynucleotide residues in the DNA in the nucleus belongs to the B-type family of conformations. However, the DNA in the nucleus may have a different furanose ring pucker than it does in solution, which may be evidence of some significant polymorphism.

A Duplex of the Oligonucleotides d(GGGGGTTTTT) and d(AAAAACCCCC) Forms an A to B Conformational Junction in Concentrated Salt Solutions

The coexistence of both A form and B form tracts and formation of an A-B junction in the oligomer d(GGGGGTTTTT).d(AAAAACCCCC) in saturated sodium chloride solution have been detected by Raman spectroscopy. The entire duplex adopts the familiar B-form conformation in aqueous solution at low salt concentrations (0.1M NaCl). In 6M NaCl the adoption of an A form is observed within the G,C tract while a B-form is maintained in the A.T tract. The experimental results indicate that two different helical forms can co-exist in a rather short oligonucleotide and that formation of an A-B junction can occur over a fairly small span of bases. This is in agreement with recent rules governing the relation between base sequence and secondary structure of DNA published from this laboratory. The conformational preferences of each of the individual oligomers d(AAAAACCCCC) and d(GGGGGTTTTT) have also been investigated. The oligomer d(AAAAACCCCC) is single stranded but some evidence for base stacking is observed at 2 degrees C. In contrast, a double stranded B-form structure characterized by wobble G-T base pairing is observed for d(GGGGGTTTTT) in 0.1M and 6M NaCl.

The infrared and Raman spectra of the duplex of d(GGTATACC) in the crystal show bands due to both the A-form and the B-form of DNA.

The deoxyoligonucleotide, d(GGTATACC), forms a duplex structure that crystallizes in the DNA A form. This has been shown by both X-ray diffraction studies and Raman spectroscopy (1,2). The presence of the DNA B form has been reported using diffuse X-ray scattering from a crystal of the closely related sequence d(GGBrUABrUACC)(3). In this paper the infrared spectrum of the d(GGTATACC) crystal is presented and curve resolution of both the Raman and IR spectra have been carried out. The percentage of A and B forms have been estimated. The %B form in the crystal has been estimated from the IR spectra to be about 15% and from Raman to be about 20%. Moreover the IR spectrum of the A conformation in the crystal is slightly different from the IR spectrum of the A conformation in polynucleotide fibers in particular in the region of the phosphate stretching vibrations and of the in-plane double bond vibrations of the bases. We show that it is feasible to obtain IR as well as Raman spectra of small crystals of oligonucleotides and that this is a good method of identifying all of the different conformations that may be in the crystal.

Base Sequence Criteria and Cartesian Coordinates For Stable B/Z and B/Z/B Junctions in Relaxed DNA

It seems increasingly evident that if the Z form of DNA exists in the genome it must exist as short sections of alternating pyrimidine-purine sequences in the midst of very long sections of B-form DNA. We have determined the minimum length of a string of alternating CG base pairs that can go into the Z form in the middle of a long B form. Self-complimentary oligomers of the form T(M)(CG)(N)A(M) were synthesized. The conformation of the resulting duplex was determined in 6M aqueous NaCl solution by Raman scattering. We have found that 12 alternating CG base pairs is the minimum length required to form a stable Z form of DNA inside of a long B form section. Only the 4 center CG base pairs go into the Z form. These 4 CG base pairs in the Z form are flanked on each side by 4 CG base pairs in a non-Z (probably B) form as well as the ..TT.. ..AA.. sequences in the B form. We propose a model of the B/Z junction in which the double helix flips directly from the B form to the Z form so that there are no base pairs in the junction. In this model the B form is nucleated in the AT base pairs on each end and is propagated into the CG base pairs in the center. This model is supported by isotopic H/D exchange experiments that shows that the H/D exchange of the non-Z form CG base pairs is highly retarded and indicates that they remain in the B form. A Thermodynamic analysis of the concentration dependence of the melting point of the duplexes in both low and high salt, supports our model and rules out the possibility of hairpin formation. The enthalpy for the formation of a B/Z junction is determined to be about +16 kcal/junction. A comparison of these results with recent results on B/Z junctions in super-coiled DNA is given. Molecular modeling calculations permit us to obtain values for the coordinates and torsional angles of the oligomers showing both B/Z and B/Z/B junctions. The Cartesian coordinates for these oligomers as well as stereo figures of these models in color are available from the authors.

THE USE OF RAMAN SPECTROSCOPY TO CHARACTERIZE DOUBLE B/Z CONFORMATIONAL JUNCTIONS IN DNA

Abstract Raman spectra of the duplex of the self-complementary oligodeoxynucleotide, d[TTCGCGCGCGAA] at various salt concentrations and temperatures have been acquired. Two methods are described which indicate that the intensities of the bands at 680 cm −1 and 625 cm −1 can be analyzed using the band assignments of Tsuboi, Nishimura and colleagues to determine the relative number of CG base pairs in the Z form. The absence of bands characteristic of AT base pairs in the Z form rules out the occurrence of the Z form in the AT portion of the duplex. From these measurements the structure of the double helical duplex in concentrated salt solutions can be described as having four of the six interior CG base pairs in Z form flanked by two CG base pairs in the junction and the two AT base pairs in a short B-form segment.

The conformation of the duplex of the dodecamer, d(CGCGAATTCGCG) as a model for DNA in the liquid state.

Abstract Detailed examination of Raman spectra from the dodecadeoxynucleotide, duplex, d(CGCGAATTCGCG), in single crystals and in aqueous solution reveal significant differences in the helical conformation exhibited in each of these states. This indicates that rules such as those of Callandino and Dickerson (Ref. 1) that relate structural parameters of the B-DNA helix to base sequence are probably not compeletly valid for DNA in solution. The conformational differences encountered in DNA in proceeding from the crystal to solution include: reduction in the A-helix like character of the C,G steps flanking the central EcoRL recognition site; redistribution of furanose conformations resulting in an increase in the C3′-endo population and reduction in the breath of the C2′-endo conformation; variation in O-P torsional angles; and changes in base stacking organization about the thymine residues. The structural differences are presumably due to the relaxation of solvent and packing constraints imposed by the crystalline environment and modulated by the sequence of the dodecamer.

Evidence for DNA polymorphism in vivo: laser Raman microsopy of chromosomes in the single eukaryotic cells--comparison with model systems

There is now definitive evidence from Raman and X-ray studies that DNA in a relaxed condition in aqueous solution is in the standard B type conformation. On the other hand, DNA in crystals, in concentrated salt solutions and bound to proteins can exist in other configurations. The question arises: what is the structure of DNA in the nucleus of a living cell where its long length must be subjected to repeated folds in order to pack it into such a small space? To answer this question we designed and built a laser Raman microscope to take the Raman spectra of DNA in the packaged state in which it occurs in nature. Spectra have been taken of the DNA in salmon sperm, squid sperm, and the nucleus of the salivary gland of drosophila melanogaster. The majority of the deoxynucleotide residues in the DNA in the nucleus belongs to the B-type family of conformations. However, the DNA in the nucleus may have a different furanose ring pucker than it does in solution.

Cartesian coordinates of DNA from Raman scattering and molecular modeling

Although a great deal of work has been done lately on unusual conformations of DNA, it is not always clear how relevant these structures are to the conformations found in the genome of living organisms. It appears that it an unusual form of DNA, such as the Z form, exists in the genome, it must exist as short sections of a specific sequence such as an alternating CG Z form sequence in the midst of very long sections of B-form DNA. To examine this problem of DNA structure, we have determined the minimum length of a string of alternating CG base pairs that can go into the Z form in the middle of a long section of B-form. Rules have been developed for determining the sequence lengths required to generate Z form in the presence of B form. Although Raman spectroscopy has been very useful as a semiquantitative indication of the conformation of proteins and nucleic acids it has not been by itself capable of giving precise structural parameters such as torsional angles or Cartesian coordinates. Recently we have shown how it is possible by a combination of Raman spectroscopy, thermodynamic analysis, hydrogen-deuterium exchange and molecular modeling to generate Cartesian coordinates for oliognucleotides in solution or in the crystal. In this paper we review the principles that lead to the actual structure determination. The possible extension of these techniques to the determination of the Cartesian coordinates for bent DNA is also discussed.