Shashank S Pataskar1 and Samir K Brahmachari*1,2
1Molecular Biophysics Unit,
Indian Institute of Science,
Bangalore-560012, India
2Functional Genomics Unit,
Centre for Biochemical Technology,
CSIR Delhi- 110007, India
*Author to whom correspondence should be addressed. Phone: 091-011-7257471,7416489;
Fax: 091-011-7257471, 091-011-7416489; E-mail: skb@cbt,res,in
Progressive myoclonus epilepsy [EPM1] is an autosomal recessive disorder caused by mutations in cystatinB [CSTB] gene which codes for a cysteine proteinase inhibitor. A majority of EPM1 alleles contain expansion of a dodecamer repeat d[CCCCGCCCCGCG]n /d[GGGGCGGGGCGC]n present 70 nucleotides upstream of the transcription start site of CSTB gene. The normal alleles contain 2 to 3 copies of this repeat while the mutant alleles contain 16 to 75 copies of this repeat. The mutant alleles with expanded repeats show reduced levels of CSTB mRNA in the blood. This study has been carried out to understand the length dependant variation in the structure of the EPM1 repeat sequence which might provide a clue about the mechanism of expansion of this repeat. Oligonucleotides were synthesized containing one, two and three copies of the repeat sequence d[GGGGCGGGGCGC]n called G12(n=1), G24(n=2), G36(n=3) and of the repeat sequence d[CCCCGCCCCGCG]n called C12(n=1),C24(n=24) and C36(n=3). The structural characterization was carried out by CD spectroscopy, UV melting, gel electrophoretic mobility on native polyacrylamide gel[PAGE], chemical modification by DEPC, DMS and P1 nuclease cleavage.
CD spectra of G12, G24 and G36 in 70mM KCl showed a positive peak at 264nm and a broad positive shoulder between 280nm and 290nm.The CD spectra for G strand oligonucleotides in 70mM NaCl showed a positive band around 290nm and a negative band around 250nm. PAGE experiments showed that all the G oligonucleotides migrate faster than their respective sizes in NaCl as well as KCl. However, G12 also showed a retarded band in the presence of NaCl. Chemical modification experiments by DMS and DEPC suggest that some of the G residues are involved in Hoogstein type of base pairing. P1 nuclease cleavage of G24 and G36 produced cuts at specific residues suggesting that these are present in the loop regions. Our studies suggest that depending on the ionic strength and repeat length G strand oligonucleotides exhibit conformational equilibrium between folded hairpin and tetraplex structure. With increase in the length of the repeat G strand oligonucleotides tend to fold back and predominantly form intramolecualr structures which involve G-G base paring of the Hoogstein type.
The C strand oligonucleotides [C12,C24 and C36] exhibited a shift in
their CD profile on lowering the pH from 7.2 to 5.0. At pH 5.0 they showed
an intense positive peak at 285nm and a negative peak at 260nm, a characteristic
CD signature of an " i-motif " structure. At pH 7.2 the C strand
oligonucleotides showed a positive peak at 278 nm and a negative peak around
260nm. PAGE experiments indicated that the C strand oligonucleotides adopt
unique, compact structures as they showed faster mobilty on polyacrylamide
gels. These studies show that C strand oligonucleotides adopt a folded back
structure similar to an intramolecular " i-motif " with an increase
in the length of the repeat. We demonstrate that both C strand as well as
G strand of EPM1 repeat sequence adopt different conformations in a length
dependant manner. Formation of these stable, intramolecularly folded structures
in both the strands could provide nucleation for the expansion of these
repeats.
J. Cromsigt1*, J. Schleucher1, J. Zdunek1, C. Hilbers2 and S. Wijmenga1
1Department of Medical Biochemistry and Biophysics,
Umeå University,
Sweden
2Department of Biophysical Chemistry,
University of Nijmegen,
The Netherlands
*Author to whom correspondence should be addressed. Phone: ++46-90-78606199;
Fax: ++46-09-136310; E-mail: jennyc@indigo.chem.umu.se
For larger biomolecules size is still a limiting factor for NMR spectroscopy. Our aim is to improve the accuracy of the structure determination of larger molecules by means of NMR with particular emphasis on RNA. We intend to achieve this by using chemical shifts as additional constraints in structure refinement and deuteration approaches. It has long been recognized that the chemical shift values may contain important structural information. Recently the reliability of chemical shift calculations of 1H nuclei in proteins has been investigated (1) and it has been shown that chemical shift derived constraints as an extra restraining force in molecular dynamics improved the accuracy of the derived structure (2). Furthermore it has been shown for DNAs that 1H chemical shifts can be calculated with good accuracy (3). We have extended the DNA study by testing the reliability of RNA chemical shift calculations. A database of 21 well-determined RNA structures was used to compare calculated and empirical chemical shifts. The results show that for RNA chemical shifts can be calculated with good accuracy (rmsd 0.24 ppm), and structural origins of chemical shifts can be explained (4,5).
When trying to extend the NMR method to larger RNA systems and to obtain higher structural detail it is essential to achieve two things: 1) narrower NMR lines, and 2) a simplified proton network. Deuteration of a part of the molecule dilutes the proton network and will lead to line narrowing. RNA molecules can be enzymatically synthesized with specific labeled NTPs, e.g. sugar protons deuterated except H2¢ and 13C/15N labels in the base. These NTPs can be obtained by enzymatic production of specifically deuterated and/or 13C-labeled riboses and attachment to a variety of 13C/15N labeled bases (6). This novel method will be employed in structural studies of the primer binding site (PBS) in Hepatitis B virus (HBV) pregenomic RNA. The primer binding, in HBV, is a key-step in the reverse transcription of the viral DNA (7). The structure of PBS, determined with the aid of specifically labeled RNA oligonucleotides, will help to understand this essential step and may provide valuable insights for the design of anti-viral drugs.
References and Footnotes
1. Ösapay, K., Theriault, Y., Wright, P., Case, D.A.,
J. Mol. Biol. 244, 183-197 (1994).
2. Kuszewski, J., Gronenborn, A.M., Clore, M., J. Magn. Reson. B107,
293-297 (1995).
3. Wijmenga, S.S., Kruithof, M., Hilbers, C.W., J. Biomol. NMR 10,
337-350 (1997).
4. Cromsigt, J.A.M.T.C., Van Buuren, B.N.M., Zdunek, J., Schleucher, J.,
Hilbers, C.W., Wijmenga, S. S., Proc. 13th ISMAR, Berlin, 132 (1998).
5. Cromsigt, J.A.M.T.C., Hilbers, C.W., Wijmenga, S. S, in preparation.
6. Tolbert, T.J., Williamson, J.R., J. Am. Chem. Soc., 119, 12100-12108
(1997).
7. Kidd, A.H., Kidd-Ljunggren, K., Nuc. Acid. Res., 24, 3295-3301
(1996).
M. Fojta1, M. Brazdova1,
H. Cernocka1, P. Pecinka1,
V. Brazda1, J. Palecek1,
J. Buzek1,
B. Vojtesek2, V. Subbaramanian3, T. M. Jovin3 and E. Palecek1
1Institute of Biophysics,
Academy of Sciences of the Czech Republic,
Kralovopolska 135, 612 65 Brno, Czech Republic
2Masaryk Memorial Cancer Institute,
656 53 Brno, Czech Republic
3Max Planck Institute for Biophysical Chemistry,
Göttingen, Germany
Wild type human tumor supressor p53 protein binds preferentially to supercoiled (sc) DNA in vitro both in the presence and absence of the p53 consensus sequence (p53CON) (1). This binding produces a ladder of retarded bands on the agarose gel. The results of DO-1 immunoblotting suggest that the bands on the blot correspond to the ethidium-stained DNA bands. Oxidation of the protein with diamide resulted in a decrease of the number of the retarded bands; nevertheless, a strong band of the p53-scDNA complex remained observable at low p53/scDNA ratios (down to 1). Increasing of the reduced p53/DNA ratio up to 15 resulted in appearance of additional retarded (well separated) bands, while oxidized p53 yielded smearing of the scDNA in the gel at the high p53/DNA ratio. Under the same conditions, no binding of oxidized p53 to p53CON in a linear DNA fragment was observed. Formation of the p53 complex with p53CON and with scDNA resulted in partial protection of the protein against oxidation by diamide and by some other oxidation agents (osmium tetroxide, hydrogen peroxide, active chlorine). In agreement with the literature (2) oxidation of p53 with diamide was irreversible and was not reverted by a ten-fold excess of DTT. On the other hand, when p53 was exposed to diamide in the presence of 0.1 mM zinc ions, binding of p53 to p53CON as well as to scDNA were restored after addition of an excess of DTT and EDTA. Other divalent cations tested (cadmium, cobalt, nickel) exhibited no such effect. We suggest that the irreversibility of p53 oxidation is due, at least in part, to the removal of intrinsic zinc from its position in the DNA binding domain after oxidation of the three cysteines to which the zinc ion is coordinated in the reduced protein.
The intensity and the number of bands of p53-scDNA complex are decreased by physiological concentrations of unchelated zinc ions (3). The binding of additional zinc ions to p53 appears much weaker than the binding of the intrinsic zinc ion in the DNA binding site of the core domain. In contrast to previously published data (4) suggesting that 0.1 mM zinc ions do not influence p53 binding to p53CON in a DNA oligonucleotide, we show that zinc efficiently inhibits binding of p53 to p53CON in DNA fragments at concentrations by one order of magnitude lower. Nickel and cobalt ions inhibit binding of p53 to scDNA and to p53CON in linear DNA fragments less efficiently than zinc. Modulation of binding of p53 to DNA by physiological concentrations of zinc might represent a new path of regulation of p53 activity in vivo.
Binding the human p53 core domain (segment 94-312) to scDNA greatly differs from that observed with the full-length p53 (5). The core domain does not posses the ability to bind strongly to many sites in scDNA regardless of the presence or absence of p53CON suggesting involvement of some other domain (probably C-terminal) in binding of the full-length p53 to scDNA. The core domain binds to p53CON in scDNA without forming the ladder of bands produced by the full-length p53 on the agarose gels and without any sign of DNA relaxation. Our studies of thermal stabilities of p53-DNA complexes show that binding of p53 to p53CON in scDNA is more stable than to other sites in scDNA not containing p53CON.
References and Footnotes
1. Palecek, E., Vlk, D., Stankova, V., Brazda, V., Vojtesek,
B., Hupp, T.R., Schaper, A. and Jovin, T.M., Oncogene 15, 2201-2209
(1997).
2. Hainaut, P. and Milner, J., Cancer Res. 53, 4469-73 (1993).
3. Palecek, E., Brazdova, M., Cernocka, H., Vlk, D., Brazda, V. and Vojtesek,
B., Oncogene in press (1999).
4. Coffer, A.I. and Knowles, P.P., Biochim. Biophys. Acta 1209, 279-285
(1994).
5. M. Brazdova, V. Brazda, J. Palecek, J. Buzek, V. Subramanian, T. M. Jovin
and E. Palecek, in preparation.
Mark J. van der Woerd1, Matthew M. Skinner1, Ying Luo1, Zvonimir Siljkovic1, Alan M. Friedman1, Shuang-yong
Xu2, John J. Pelletier2,
Diaqing Liao3, Patrick P. Dennis4
1Dept. of Biological Sciences,
Purdue University, W. Lafayette, IN 47907
2New England Biolabs, Beverly, MA 01915
3Univ. de Sherbrooke,
Sherbrooke, Quebec, J1H 5N4
4Univ. of British Columbia,
Vancouver, BC V6T 1Z3.
As part of our long-term effort to understand protein-nucleic acid interactions under extreme conditions and to exploit the favorable properties of thermophilic proteins in structural studies, we have recently determined the crystal structure of two nucleic-acid binding proteins from thermophiles. The first is the nusG homologue from the extreme thermophile, Thermotoga maritima, a protein that shows extremely cooperative, non-specific binding to nucleic acids. The second is the thermostable restriction endonuclease BsoBI from Bacillus stearothermophilus. This enzyme is a thermostable isoschizomer and also a close homologue (55% identity) to the well-known mesophilic restriction enzyme AvaI.
In E. coli, NusG is an accessory transcription factor that reduces pausing by RNA polymerase and yet also enhances the efficiency of rho-dependent termination. The nusG homologue from T. maritima codes for a much larger protein (40 kD versus 21 kD for the E. coli protein) which also has nucleic-acid binding activities not detected in E. coli NusG. Sequence comparisons and proteolytic analysis suggested that T. maritima NusG forms three domains. We have crystallized and determined the structure of the two N-terminal domains of the T. maritima NusG to a resolution of 2.0Å (schematic below). This part of the sequence is marked by a large 171-residue insertion into sequences common to all nusG homologues. The two-domain fragment crystallizes as a dimer that forms an extremely elongated molecule, 200Å in length. The two domains are dominated by beta strand secondary structure with some structural similarities to other nucleic-acid binding proteins. The common nusG sequences form one alpha/beta domain of discontinuous sequence, while the 171-residue insertion forms a separate domain, itself composed of three beta sandwich subdomains. The dimeric interface is isologous and occurs across one of the subdomains.
The nusG sequences from several completely sequenced genomes form a cluster of orthologous genes (COG #250) in the genetic taxonomy of Koonin. Employing the nusG homologue from T. maritima has allowed us to obtain structural information for this family of proteins, despite the lack of success in previous attempts to crystallize the E. coli NusG. The primitive state of biochemical and genetic studies in T. maritima requires us at present though to deduce function from structure, a situation common to much of structural genomics. Models of nucleic acid binding to T. maritima NusG that are suggested by the structure will be discussed.
The thermophilic BsoBI restriction endonuclease recognizes the degenerate sequence CPyCGPuG and cleaves after the first C. Its rare combination of thermostability and favorable kinetic properties has been exploited in the development of the strand displacement amplification reaction, an isothermal alternative to PCR for the amplification of nucleic acids for diagnostic purposes. The close sequence homology between BsoBI and AvaI should allow the identification of a relatively small number of substitutions that are the source of its thermostability and its ability to bind and cleave DNA at elevated temperatures.
We have determined the crystal structure of the restriction endonuclease
BsoBI complexed to a cognate DNA target to 1.7Å
resolution (schematic
below). Each monomer of the dimeric BsoBI contains two domains that together
completely encircle the DNA. One domain contains a modified version of the
common restriction endonuclease alpha/beta fold. The dimeric interface of
this domain is significantly different and much less extensive, however,
in BsoBI than in the other enzymes. The second domain is largely alpha-helical
without close homologues in the Dali/FSSP database. Connected to the first
domain by two long alpha-helices, the second domain extends above the DNA
binding pocket and makes a second dimeric interface on the opposite face
of the DNA. The DNA is essentially straight although somewhat extended and
underwound. DNA interactions are made by all three structural elements of
the protein. Extensive water-mediated interactions (not yet fully characterized)
to both bases and backbone are also seen. The mechanisms by which the protein
binds and encircles the DNA are under investigation. Significant conformational
changes or assembly of monomers around the DNA will be required.
J.-Ph. Demaret1 and M. Gueron*2
1L.P.B.C.,
Universite Pierre et Marie Curie,
4 place Jussieu, 75252
Paris cedex 05, France
2Groupe de biophysique de l'Ecole polytechnique
et de l'UMR 7643 du CNRS,
Ecole polytechnique,
91128 Palaiseau, France
We showed recently that the transition of poly[d(G-C)].poly[d(G-C)] from Z-DNA to B-DNA as the ionic strength is reduced ([NaCl] < 2.25 M/L, or correspondingly [MgCl2] < 0.7 M/L), can be ascribed to the lesser electrostatic free energy of the B form, due to better immersion of the phosphates in the solution. This property was incorporated in cylindrical models of B-DNA and Z-DNA which were analyzed by Poisson-Boltzmann theory. The results are insensitive to details of the models, and in fair agreement with experiment.
In contrast to poly[d(G-C)].poly[d(G-C)], the Z form of the poly[d(G-m5C)] duplex is stabilized by very small concentrations of magnesium. We now show that this phenomenon is easily explained by the same electrostatic theory, without any adjustable parameter. The very different responses to magnesium of the methylated and non-methylated polymers stem from a modest change in the non-electrostatic component of the free energy difference between the Z and B forms (as measured independently in supercoiled plasmids), and not from any stereo-specific interaction between DNA and the cation.
The theory also explains the effect of micromolar concentrations of trivalent cobalt hexammine on the B-Z transition, and it provides a framework for describing the influence of temperature and of solvent changes.
Hence, in the case of the B-Z transition as in others (e. g. the folding of tRNA or of the hammerhead ribozyme), the effect of ions on nucleic acid structure is mediated primarily by electrostatic, non-specific interactions which are well described in a simple electrostatic theory which ignores atomic details, charge correlations and solvent structure.
The weak influence of specific ion -nucleic acid interactions on nucleic
acid structure may be a general phenomenon, since it appears that convincing
counter-examples are lacking.
Anh Tuân Phan, Jean-Louis Leroy and Maurice Guéron
Groupe de Biophysique
de l'Ecole Polytechnique et de l'UMR 7643 du CNRS,
91128 Palaiseau, France
NMR spectroscopy can provide detailed information on the association between DNA and water in solution. An Overhauser effect between water and DNA protons reveals and locates bound water molecules. If the residence time is in the suitable range, the zero-NOE condition, which may be achieved by varying the proton NMR frequency and/or the temperature, provides an accurate value of the residence time (e.g. 0.356 ns at the proton frequency of 500 MHz) (1). Using a variant of this method, the zero-(off-resonance ROE), the change in NMR frequency or temperature becomes unnecessary.
In the minor groove of the B'-DNA duplex of d(CGCGAATTCGCG), a water proton next to the central adenine (6, underlined), has a residence time of 0.6 ns at 10 °C. The residence time of the water next to A5 is slightly shorter. The distinct difference suggests non-cooperative departure of the two molecules. These residence times are longer than those in the minor groove of the regular B-DNA sequence d(CGCGATCGCG) by only a factor of 2. This suggests that the spine of hydration is perhaps not a major stabilizer of the B'-DNA structure as compared with B-DNA.
Major groove residence times of these and other B and B' duplexes are similar, and shorter than minor groove times. The shortest times are only ten times less than the longest times observed in any minor groove at the same temperature. Thus, the difference in the residence times of reputedly fast and slow exchanging water molecules bound to DNA is rather small.
We have also studied the water residence times in a triple helix, in a G-tetrad and in i-motif structures. The residence time is moderately enhanced in narrow grooves and nooks. It could perhaps be used as a probe of surface topography.
The approach just described may be applied to any protonated DNA ligand, and in particular to cations such as ammonium, cobalt hexammine, or hydrated magnesium. Since their lifetime within the cation is much longer than nanoseconds, the protons may be used as probes of the location and residence time of cations interacting with DNA.
Previously, 15N ammonium ions have been reported in the minor groove of B' DNA (2). We detect them also in B DNA. In both cases we find residence times in the range of nanoseconds, comparable to those mentioned above for water molecules. Again, they are slightly longer in B' DNA than in B DNA. The similar behavior of water and ions brings to mind a recent X-ray study (3) indicating that a fraction of the sites of the "spine of hydration" of B'-DNA may be occupied by sodium rather than water. We find that the intensities of DNA/water proton NOEs are compatible with partial occupancy of the water sites in the minor groove of B' DNA, without violation of the minimum Van der Waals distance between protons.
Upon addition of magnesium, new sites for bound water are observed in the major groove of the B' DNA duplex of d(A5T5). The residence time, which is presumably that of the partially or totally hydrated ion, is longer than 0.5 nanoseconds at 15 °C. We find no such site in the major or minor groove of the B-DNA sequence d(CGCGATCGCG).
References and Footnotes
1. Phan, A.T., Leroy, J.L. & Guéron, M., J.
Mol. Biol. 286, 505-519 (1999).
2. Feigon J., XVIIIth International Conference on Magnetic Resonance
in Biological Systems, Tokyo, p.39 (1998).
3. Shui, X., McFail-Isom, L., Hu, G. G. & Williams, L. D., Biochemistry
37, 8341-8355 (1998).
Surjit B. Dixit and B. Jayaram
Department of Chemistry,
Indian Institute of Technology,
Hauz Khas, New Delhi-110016, India
Hydrogen bonds have been accredited with a major role historically, in the formation and stabilization of biomolecular structures. The formation of hydrogen bonds in aqueous medium involves not only favorable interactions of the donor and acceptor functional groups but also a loss of interactions of these groups with the solvent water. We carried out extensive molecular dynamics simulations on the AT base pairs in chloroform and water at 1 atm and 298 K in a [T,P,N] ensemble and developed a post facto free energy profile for hydrogen bond formation as a function of the distance between the bases. Hydrogen bond formation was disfavoured in aqueous medium due to the dominant desolvation expense but favoured in chloroform. We further performed a series of calculations on the free energy of hydrogen bond formation at the interface of 40 protein-DNA complexes. Over all, our results support the emerging view that hydrogen bonds in particular and electrostatics in general contribute unfavourably to the thermodynamics of complex formation in aqueous medium. Hydrogen bonding interactions however do act to stabilize the complex once it is formed.
Neocles B. Leontis*
Chemistry Department,
Bowling Green State University,
Bowling Green, OH 43403
*For author correspondence. Phone: 419-372-8663; Fax: 419-372-9809; E-mail:
leontis@bgnet.bgsu.edu
Nucleic acid bases interact by stacking or abutting edge-to-edge. The, edge-to-edge interactions, mediated by electrostatically driven hydrogen-bonding between complementary arrays of negatively and positively polarized. Nucleic acid bases are aromatic heterocycles with a large proportion of heteroatoms. This feature generates multiple modes of H-bonding interactions. We will present a coherent, geometrically based organization of the available crystallographic data of RNA edge-to-edge base-base interactions and apply this to construct a coherent library of isosteric pairings that can substitute for each other in homologous RNA molecules. The underlying assumption is that the three-dimensional structures of homologous RNA molecules are more strongly conserved than their individual sequences. Crystallographic data, therefore, become even more useful when correlated with comparative sequence analysis, for the simple reason that isosteric pairs will substitute for each other in conserved regions or motifs. Conversely, covariation data contains information that can potentially identify bases involved in tertiary interactions and even indicate the most likely pairing geometry. Our goal in organizing the data in this way is therefore to facilitate prediction of RNA tertiary structure from sequence.
E.E. Minyat*1, A.D. Beniaminov1, N.G. Dolinnaya2 and V.I.
Ivanov1
1Engelhardt Institute of Molecular Biology,
Russian Academy of Sciences,
32 Vavilov str.,
Moscow 117984, Russia
2Belozersky Inst. of Phys.-Chem. Biology,
Moscow 119899, Russia
*Author to whom correspondence should be addressed. Phone: 7-095-135-1255;
Fax: 7-095-135-1405; Email: chrom@imb.imb.ac.ru
Folding nucleic acids leading to formation of more complex, tertiary, structures requires detail understanding the principles controlling the DNA and RNA structures. We have recently proposed a new type of spatial packing for a polynucleotide chain, the Slipped-Loop Structure - SLS (1,2). The existence of SLS was proved experimentally on the synthetic oligonucleotides by chemical modification method and 'H NMR technics (3). However, such a structure can be present in different isomeric forms in equilibrium.To avoid that situation we designed the circular oligonucleotides able to adopt only proper SLS. Using chemical ligation and enzymatic cyclization we demonstrated that this strategy allowed to make a choice of a unic SLS isomer, to prove the existence of the SLS folding and thereby to demonstrate a novel possibility for polynucleotide chain folding.
Supported by RFFI grants 98-04-49101 and 96-15-98093
References and Footnotes
1. Gorgoshidze, M.Z. Minyat, E.E., Gorin, A.A., Demchuk,
E.Ya., Farutin, V.A. and Ivanov, V.I., Molecular Biology (English translation)
26, 832-838 (1992).
2. Ulyanov, N.B., Bishop, K.D., Ivanov, V.I. and James, T.L., NAR 22,
4242-4249 (1994).
3. Ulyanov, N.B., Ivanov, V.I., Minyat, E.E., Khomyakova, E.B., Petrova,
M.V., Lesiak, K.and James, T.L., Biochemistry 37, 12715-12726 (1998).
A.K. Nagaich1,2, V.B. Zhurkin3, E. Appella2 and R.E. Harrington1
1Department of Microbiology,
Arizona State University,
Tempe, AZ 85287-2701
2Laboratory of Cell Biology,
3Laboratory of Experimental and Computational
Biology,
NCI, National Institutes of Health,
Bethesda, MD 20892 USA
DNA binding activity of p53 is crucial for its tumor suppressor function. Our recent studies have shown that four molecules of the DNA binding domain of human p53 (p53DBD) bind the response elements with high cooperativity and bend the DNA1-3. Using A-tract phasing experiments, we find significant differences between the bending and twisting of DNA by p53DBD and by full length human wild type p53 (WT p53). Our data show that four subunits of p53DBD bend the DNA by 32°-36° whereas WT p53 bends it by 51°-57° 4. The directionality of bending is consistent with major groove bends at the two pentamer junctions in the consensus DNA response element. More sophisticated phasing analyses also demonstrate that p53DBD and WT p53 overtwist the DNA response element by ~35° and ~70° respectively. These results are in accord with molecular modeling studies of the tetrameric complex. The DNA can assume a range of conformations resulting from correlated changes in bend and twist angles such that the p53-DNA tetrameric complex is stabilized by DNA overtwisting and bending toward the major groove at the CATG tetramers 5. Overall, the four p53 moieties are placed laterally in a staggered array on the external side of the DNA loop and have numerous inter-protein interactions that increase the stability and cooperativity of binding. Notably, the DNA deformations detected upon p53 binding in solution are consistent with the anisotropic bending and overtwisting of DNA observed in the nucleosomes. To further explore the DNA binding and bending behavior of p53, we have carried out binding studies of p53 with the response element DNA reconstituted in the nucleosomes. Novel findings regarding the rotational and translational positioning of p53 response elements with respect to nucleosomal dyad, binding of p53 and its possible functional implications in chromatin will be discussed.
References and Footnotes
1. Balagurumoorthy, P., Sakamoto, H., Lewis, M. S., Zambrano,
N., Clore, G.M., Gronenborn, A. M., Appella, E. & Harrington, R. E.,
Proc. Natl. Acad. Sci. USA 92, 8591-8595 (1995).
2. Nagaich, A. K., Zhurkin, V. B., Sakamoto, H., Gorin, A. A., Clore, G.
M., Gronenborn, A. M., Appella, E. & Harrington, R. E., J. Biol.
Chem. 272, 14830-14841 (1997).
3. Nagaich, A. K., Appella, E. & Harrington, R. E., J. Biol. Chem.
272, 14842-14849 (1997).
4. Nagaich, A.K., Zhurkin, V.B., Durell, S.R., Jernigan, R.L., Appella,
E. and Harrington, R.E., Proc. Natl. Acad. Sci. USA, (in press) (1999).
5. Durell, S. R., Appella, E., Nagaich, A. K., Harrington, R. E., Jernigan,
R. L. & Zhurkin, V. B. in Structure, Motion, Interaction and Expression
of Biological Macromolecules. Proceedings of the Tenth Conversation,
eds. Sarma, R. H. & Sarma, M. H. (Adenine Press, Schenectady, NY), pp.
277-295 (1998).
G.Natarajan*1, R.Malathi1
and Eggehard Holler*2
1Dept. of Genetics,
Dr.ALM Post Grad.Inst.Basic Med.Sci,
Taramani, Madras-600 113, India
2Inst. f. Biophysik. u. Physik. Biochem.,
Universität Regensburg,
D-93040, Regensburg, Germany
*Correspondence may be addressed to either author.
G. Natarajan Phone: 044-492 5051; Fax: 044-492 6709; E-mail: natarajanganesan@hotmail.com
E. Holler Phone:(00-49-941) 943 3030; Fax : (00-49-941)9432813; E-mail:
eggehard.holler@biologie.uni-regensburg.de
Two theories exist to explain the molecular mechanism of carboplatin (second generation analogue of the popular anti-tumor drug Cisplatin) with DNA
1) Aquation hypothesis or the "like cisplatin" hypothesis
2) Activation hypothesis or the "unlike cisplatin" hypothesis
of which the former is more accepted owing to the similarity of the leaving groups with its predecessor cisplatin. The latter hypothesis envisages a biologically activated mechanism to release the active Pt2+ before exerting its biological activity.
In the present study, we report the changes in the DNA binding properties of Carboplatin in the presence of nucleophiles viz. methionine, glutathione, thiourea and increased pH(9.0), and cytoplasmic extracts of MCF-7 cell lines. The results have been compared with cisplatin and other novel platinum (II) complexes, viz. Polymalate-Pt(PMA-Pt), Malate-Pt(Mal-Pt)and Succinate-Pt(Succ-Pt) complexes which have similar leaving groups i.e. closed dicarboxylate, with closed dicarboxylate around the leaving arm of Pt(II). It could be clearly observed that carboplatin showed increased DNA binding activity in the presence of very same agents that inhibited the activity of cisplatin and, more interestingly, the other 'carboplatin like' complexes. The resultant changes in the morphology of MCF-7 cell lines on treatment with carboplatin have also been observed to be in contrast with that of cisplatin and the other Pt(II) complexes.
We propose a chemically mediated (nucleophilic attack) release of CBDCA
moiety by S-containing compounds in the cellular metabolic mileu before
reacting with its target sequences. The variation in the aquation kinetics
possibly confers a new mode of reactivity to the Pt(II) complex thereby
forming altogether different growth limiting adducts with DNA. There is
probably much more to the role of the non-square planar moiety, viz. CBDCA,
than meets the eye in conferring to the reduced side effects during cancer
treatment.
Taro Nishinaka1,2, Shigeyuki Yokoyama2,3 and Takehiko Shibata1
1Cellular & Molecular Biology Laboratory,
The Institute of Physical and Chemical Research (RIKEN),
Wako, Saitama 351-0198, Japan
2Department of Biophysics and Biochemistry,
Graduate School of Science,
The University of Tokyo,
Bunkyo, Tokyo 113, Japan
3Cellular Signaling Laboratory,
The Institute of Physical and Chemical Research (RIKEN),
Wako, Saitama 351-0198, Japan
By NMR analysis, we previously reported a novel deoxyribose-base stacking interaction between adjacent residues in the single-stranded DNA within the filament made by Escherichia coli RecA protein. In the RecA-bound form, the 2¢-methylene moiety of each deoxyribose is located above the base of the next residue in place of the normal base-base stacking. The DNA was extended by approximately 1.5 times compared with the B-form DNA. Based on this structure, we have formulated the structure of duplex DNA within filaments formed by RecA protein and its homologs. Two types of the structure (ATP-form & ADP-form) will be presented. In each DNA structure, interconversion of the sugar pucker between the N-type and the S-type rotates bases horizontally, while maintaining the deoxyribose-base stacking interaction. We propose that this base-rotation enables base-pair switching between double-stranded DNA and single-stranded DNA to take place, facilitating homologous pairing and strand exchange.
Witold R. Rudnicki*, Grzegorz Bakalarski and Bogdan Lesyng
Interdisciplinary Centre for Mathematical and Computational Modeling,
Warsaw University,
Pawiaw University Centre for Mathematica,
Pawiñskiego 5A,
02-106, Warsaw, Poland
*Author to whom correspondence should be addressed. Phone: +48 22 8749 100;
Fax: +48 22 8749 115; E-mail: W.Rudnicki@icm.edu.pl
Conventional, microscopic molecular dynamics (MD) methods are used to simulate dynamical properties of biopolymers in a short time scale, usually in the range from 10-11s to 10-9s, while biologically interesting phenomena may occur in the time scale of 10-9s to 10-3s or longer. This makes the direct microscopic MD simulations of such phenomena difficult. In some cases special techniques can be applied to get results of practical relevance, see e.g. (1-2). Increasing the computing power is not sufficient to solve the problem and therefore development of new models and theories, that would allow for significantly longer simulations, is of particular importance.
We formulated a general model for molecular dynamics simulations with objects of different scale and properties, e.e. atoms, pseudoatoms, rigid and pseudo-elastic bodies, with external and internal degrees of freedom. This approach leads both to a reduction in the number of degrees of freedom and to an increase in the integration time-step. In the case of DNA/RNA furanose rings (3), we achieved a twenty-fold increase in the integration time-step and a fifty-fold reduction in the number of degrees of freedom. Reduction of the number of degrees of freedom requires effective interaction potentials. In most cases one uses potentials of mean force (PMF), see e.g. (4). In this study we applied a PMF, which was created based on microscopic MD simulations with atom-atom potentials tuned for simulations of nucleic acids, see (5-6).
The equations of motion for the molecular system are obtained from the Lagrange function. Rigid body motion equations are solved using quaternion parameters. We call the model Lagrangian and Quaternion Molecular Dynamics (LQMD). We present the theoretical derivation of the LQMD equations, their implementation into a functional code and results of the simulations. The open structure of the LQMD model allows for applying objects of varying resolution for different subunits of the system. In particular a part of the system can be described using the all-atom model, while the rest of the molecule can be modeled using various types of reduced representations.
The LQMD model has been applied to simulations of the dynamics of nucleic acids. Stability of the developed algorithms for various time-steps has been tested. The total energy, momentum and angular momentum are conserved for the time-steps up to 20 fs.
This research was supported by KBN (8T11F01616)
References and Footnotes
1 Lesyng, B. and McCammon, J., Pharmac. Ther. 60,
149-167 (1993).
2 Straatsma, T. and McCammon, J., Annual Review of Physical Chemistry,
43, 407-435 (1992).
3 Rudnicki, W., Lesyng, B., and Harvey, S., Biopolymers 34, 383-392
(1994).
4. Kuczera, K., J. Comp. Chem. 17, 1726-49 (1996).
5. Rudnicki, W. and Lesyng, B., Computers Chem. 19, 253-258 (1995).
6. Maciejczyk, M., Rudnicki, W., and Lesyng, B. in preparation.
Bernd N.M. van Buuren1*, Franc J.J. Overmars2, Johannes H. Ippel1, Cornelis
Altona2, Harald Schwalbe3,
Christian Griesinger3, Juergen Schleucher1 and Sybren S. Wijmeng1
1Department of Medical Biochemistry and Biophysics,
Umeå University,
90735 Umeå, Sweden
2Leiden Institute of Chemistry,
Gorlaeus Laboratories,
P.O. Box 9502,
NL-2300 RA Leiden, The Netherlands
3Institute for Organic Chemistry,
University of Frankfurt,
Marie-Curie Str. 11,
60439 Frankfurt, Germany
*Author to whom correspondence should be addressed. Phone: ++44-90-7866199;
E-mail: bernd@indigo.chem.umu.se
Branched DNA species are commonly postulated as intermediates in DNA rearrangements. DNA Three- (TWJ) and Four-Way junctions (FWJ) are involved in genetic recombination processes. TWJs are observed in recombination processes involving phages and FWJs are the proposed intermediate in homologous recombination. In both cases, enzymes must interact specifically with the DNA structure to bring about the recombination event. These proteins recognize their DNA substrates at the level of tertiary structure rather then at the level of secondary structure. A considerable amount of structural knowledge has been derived for TWJs and FWJs by a variety of biophysical methods including NMR, however until now no detailed structure has been established. Here we present the results of our NMR structural studies on DNA Three- and Four-Way junctions.
We have derived the solution structure of a DNA TWJ containing two unpaired thymidines, this TWJ (TWJ1) has been shown to have a high preference for the A/B stacked conformer (1). 100 structures were calculated via a torsion angle dynamics protocol. The 20 lowest energy structures all converted to a single conformation. To asses the accuracy of these structures 1H chemical shifts were calculated (2,3) and compared with the experimental shifts. This showed a very low (< 0.2) chemical shift RMSD indicating that the structures fulfill the experimental observations very well.
Similar studies on a DNA FWJ (J6), with an AAGG junction site (see (4), show that J6 has a high preference for an A/D stack when Mg2+ ions are present. This high preference ((80%) is confirmed by X-filtered NMR experiments using labeled nucleotides at the junction site. The metal binding site of nucleic acid structures can be studied by NMR using cobalt(III)hexammine (5). We used this approach to determine the metal binding site of J6. From our NMR data, we could conclude that the stacking preference of J6 is not influenced by the cobalt(III)hexammine. In NOESY experiments we observe contacts between J6 and the amine protons of the cobalt complex. Structural implications will be discussed
References and Footnotes
1. Overmars, F.J.J., Pikkemaat, J.A. van den Elst, H.,
van Boom, J.H. and Altona, C.A., J. Mol. Biol. 255, 702-713 (1996).
2. Wijmenga, S.S., Kruithof, M. And Hilbers, C.W., J. Biomol. NMR 10,
337-350 (1998).
3. Wijmenga S.S. and van Buuren, B.N.M., Prog. NMR Spectr. 32, 287-387
(1998).
4. Altona, C.A., J. Mol. Biol. 263, 568-581 (1996).
5. Kieft and Tinoco Jr, I., Structure 5, 713 (1997).
Kissinger Goldman, Meghan Lembeck, and James S. Godde*
Department of Biology,
The City University of New York,
Brooklyn College,
Brooklyn, NY 11210
*Author to whom correspondence should be addressed. Phone: (718) 951-5723;
Fax: (718) 951-5642; E-mail: jgodde@brooklyn.cuny.edu.
We have been investigating the formation of interstrand crosslinks in the human Fragile X Mental Retardation Gene (FMR1) by mitomycin C, hereafter MC. This antitumor antibiotic creates covalent crosslinks between the N-2 positions of guanines upon bifunctional activation, targeting the CpG dinucleotide as its exclusive site of interstrand crosslinking. This activity has been demonstrated to be further enhanced if the cytosine is methylated (1).
We chose the FMR1 system for these studies since the disease-associated gene results from both an expansion of the (CGG)n repeats beyond the normal n=6-60 as well as an accompanying hypermethylation of this sequence. We do find a significant enhancement in mitomycin-mediated crosslinking in the FMR1 gene upon cytosine methylation, well beyond the predicted 2-3 fold enhancement due to methylation effects alone (2).
The positions of both the precursor monofunctional adducts of MC as well as the positions of final crosslinks have been mapped in our laboratory and are consistent with the formation of a DNA quadruplex structure within this gene following hypermethylation. MC adduct formation is actually seen to decrease within the CGG repeats themselves, relative to other nearby CpG sites, suggesting that the guanines in question are involved in the G-tetrad structures know to stabilize quadruplex regions. The enhancement in overall MC crosslinking maps to the final CGG trinucleotide, located 3 of the G-rich repetitive region, this may indicate a region of DNA structural perturbation required to form a quadruplex. We expect that this means of investigation may enable the direct detection of quadruplex regions associated with CGG repeats where heretofore this was not possible.
This research was supported by grants from The City University of New York PSC-CUNY Research Award Program and CUNY Collaborative Incentive Grant Program.
References and Footnotes
1. Millard, J.T., Beachy, T.M., Biochemistry 32,
12850-12856 (1993).
2. Johnson, W.S., He, Q.Y., Tomasz, M., Bioorg. Med. Chem. 3, 851-860
(1995).
Sergei A. Streltsov, Larissa P. Martinkina and Yuri Yu. Vengerov
Engelhardt Institute of Molecular Biology,
Russian Academy of Sciences,
Vavilov str., 32,
117984 Moscow, Russia
The interaction of tRNA with trivaline dansyl hydrazide trifluoroacetate (DHTV) has been studied. The shape of the curves of fluorimetric titration of tRNA with DHTV and vice versa can be explained only by formation of DHTV dimers on tRNA molecules, and subsequent association of DHTV-saturated tRNA molecules with each other. The ability of tRNA molecules to form concatemers in solution in the presence of DHTV has been demonstrated by electron microscopy. Electron microscopy of the tRNA-DHTV complexes stained with uranyl acetate revealed flexible rods 6-7 nm thick and up to several micrometers long. Previously (1) it was shown that pdGTT, rAAG, rCUU assemble in thin flexible rods up to several hundred nanometers long in the presence of DHTV. These structures were formed by trinucleotides in such a way that addition of condensing agents caused covalent binding of the trinucleotides into chains containing up to 12 nucleotides (2). We suppose a common model for concatemerization of tRNA and trinucleotide molecules in the presence of DHTV.
References and Footnotes
1. Streltsov, S.A., Martinkina, L.P., Khorlin, A.A., Florentiev,
V.L., Vengerov, Yu.Yu., Zhenodarova, S.M., Sedelnikova, E.A. and Smolyaninova,
O.A., J. Biomol. Struct. Dynam. 11, 1403-1415 (1994).
2. Streltsov, S.A., Khorlin, A.A., Victorova, L.S., Kochetkova, S.V., Tsilevich,
T.L. and Florentiev, V.L., FEBS Letters 298, 57-60 (1992).
Vadim V. Demidov1, Konstantin I. Izvolsky1, Peter E. Nielsen2 and
Maxim D. Frank-Kamenetskii1
1Center for Advanced Biotechnology,
Boston University,
36 Cummington Street,
Boston, MA 02215
2Center for Biomolecular Recognition,
Copenhagen University,
Blegdamsvej 3c,
Copenhagen N, DK-2200, Denmark
PNA-assisted rare cleavage (PARC) is a promising tool for structural analysis of complex genomes (1,2). This assay makes it possible to convert common restriction enzymes into infrequent DNA cutters. The PARC strategy takes advantage of highly stable and sequence-specific complexes formed by special PNA constructs with double-stranded (ds) DNA for protection of few DNA sites from enzymatic methylation with their subsequent cleavage by restriction enzyme. The strategy relies on so-called PNA invaders that selectively bind dsDNA via strand displacement.
On the basis of the PARC strategy we have developed a method of segregation of yeast artificial chromosomes (YACs) from host chromosomes with similar lengths. YACs are useful vectors to clone and to transfer large fragments of alien (e.g. human) DNA and are very similar to endogenous yeast chromosomes in length and general structure. YACs are commonly isolated by pulsed-field gel electrophoresis (PFGE) but YAC analysis is often complicated by contamination with host chromosomes because of inability to separate them electrophoretically.
The PARC-based method of YACs purification during the PFGE separation uses cationic homopyrimidine bis-PNAs as invaders to selectively cut at designated sites those host chromosomes, which comigrate with the desired YAC. Electrophoretic removal of chromosome fragments yields convenient PFGE "windows" for the YAC isolation. M.Hha I/R.Hae II pair of enzymes in conjunction with appropriate bis-PNA invaders provides with a set of unique cutters per each chromosome. Using these cutters we have selectively eliminated 9 of 16 S.cerevisiae chromosomes from the PFGE pattern. The method was successfully applied to segregate two 'human' YACs from yeast chromosomes II and XIV (3). Due to high selectivity of the PARC method, both YACs remained virtually intact.
By choosing an appropriate enzymatic pair, it is possible to adjust the recognition specificity of PARC cutters in such a way that they can also be used for 'blind' rare cutting DNA with unknown sequences. This approach was used by us for infrequent fragmentation of 'human' mega-YAC and yeast genomic DNA.
Design of a new generation of PNAs capable of sequence-universal double-duplex invasion into dsDNA has been proposed (4). Our preliminary data on rare cleavage of adenoviral DNA demonstrate that application of suchlike neoteric PNAs as invaders may lift sequence limitations in the PARC strategy imposed by all-pyrimidine character of bis-PNAs used before (5).
References and Footnotes
1. Demidov, V.V. & Frank-Kamenetskii, M.D., PNA
directed genome rare cutting. In PNA: Protocols and Applications, P.E.
Nielsen & M. Egholm, eds. Horizon Scientific Press, Wymondham, England
(1999).
2. Frank-Kamenetskii, M.D., Triplexes and biotechnology. In Triple Helix
Forming Oligonucleotides, C. Malvy & A. Harel-Bellan, eds. Kluwer
Academic Publishers, Norwell, USA (1999).
3. Izvolsky, K.I., Demidov, V.V., Bukanov, N.O. & Frank-Kamenetskii,
M.D., Yeast artificial chromosome segregation from host chromosomes with
similar lengths, Nucleic Acids Res. 26, 5011-5012 (1998).
4. Nielsen, P.E., Design of sequence-specific DNA-binding ligands, Chem.
Eur. J. 3, 505-508 (1997).
5. This work was supported by NIH.
A.L. Mikheikin, A.L. Zhuze and A.S. Zasedatelev
Engelhardt Institute of Molecular Biology,
RAS,
117984 Moscow, Russia
A correlation was found between the experimental data on the affinity of ligands to the DNA minor groove and their hydrophobicity. To learn more about the contribution of such factors as hydrophobicity, charge distribution, and H-bonding capability, the binding of two series of ligands to the DNA minor groove was investigated theoretically and by optical methods (UV, circular dichroism, flow linear dichroism, and fluorescence):
I. Several analogs of dye Hoechst 33258 with different groups at the para-position of the phenyl ring (Figure 1) were synthesized. The compounds with amino, N,N-dimethylamino, and hydroxy groups show high affinity to DNA, and the compound with a nitro group is weakly affine. No correlation was found between the charge-distributing properties of attached groups and the affinity of the corresponding ligands to the DNA minor groove.
Figure 1
II. Binding to DNA of four compounds: 1,1',3,3,3',3'-hexamethylindocarbocyanine
(I), pinacyanol chloride (II), 3,3'-diethylthiacarbocyanine (III) and 3,3'-diethyloxacarbocyanine
(IV) (Figure 2) was investigated (ligands enumerated in decreasing affinity
to DNA). Compounds III and IV can form H-bonds as acceptors whereas compounds
I and II can not. This feature does not in
fluence the ligand binding
in the minor groove.
Figure 2
The hydrophobicity of both series of compounds was estimated experimentally using octanol/water distribution. The affinity to the minor groove was found to correlate with the octanol/water partition coefficient, testifying to the important role of ligand hydrophobicity.
A new approach to computing the interaction energy was proposed, taking into account the ligand hydrophobicity. The binding energy was obtained as the difference between the energies of the ligandminor groove and ligandwater interactions. This new procedure for the computation of the binding energy by molecular mechanics method was applied to antibiotic distamycin A, its furancarboxamide analog (1), fluorescent dyes DAPI, Hoechst 33258, its oligophthalimide analog (2), and also to compounds used in this work. The computing results were in qualitative agreement with the experimental data. These studies show that, in addition to the previously known factors (the ligand molecule should be isogeometrical to the DNA minor groove and capable to form H-bonds as donor with DNA bases in the DNAligand complex), hydrophobicity appeared to be a significant property determining the specificity of the ligand molecule to the DNA minor groove. Using this approach, new structures of DNA-specific compounds were suggested and found to be capable to bind in the DNA minor groove.
Supported by RFBR grants No. 98-03-32949 and No. 96-15-98093.
References and Footnotes
1. Mikheikin A.L. et. al., Molec. Biol. (Moscow, Engl.
trans.) 31, 854-861 (1997).
2. Salmanova D.V. et. al., Molec. Biol. (Moscow, Engl. trans.) 29,
491-498 (1995).
G. Ertem, K. J. Prabahar, P. C. Joshi and J. P. Ferris
Department of Chemistry,
RPI,
Troy, New York 12180
The condensation of RNA monomers to RNA is one of the basic tenets of the RNA world scenario for the origin of life (1). We discovered that montmorillonite clay catalyzes the condensation of activated mononucleotides to oligomers in pH 8 aqueous solution at room temperature.
It is postulated that in the RNA world scenario that sequence information in the RNA was preserved by template-directed synthesis. This proposal was tested by investigation of the synthesis of oliogo(G)s on the heterogeneous oligomers formed by clay mineral catalysis. Oligo(C) templates which contain mainly 2',5'-links together with small amounts of 3',5'-links, cyclic oligomers and pyrophosphate links, directed the synthesis of the complementary oligo(G)s (3). This experiment demonstrated that the information content of the RNA formed by prebiotic processes could have been maintained by template-directed synthesis on the primitive Earth.
Another key aspect of life in the RNA world is the catalysis of chemical processes by RNA (ribozymes). It is unlikely that RNA 6-15 monomer units in length, formed by montmorillonite-catalyzed condensation of activated monomers, would exhibit catalytic activity. It is possible to make oligo(A)s as long as 50 mers by adding activated monomer daily to a decameric primer for 14 days (4). It is proposed that an RNA containing 30-60 units would have been long enough to have catalyzed the reactions of other RNA molecules (5) and would have replicated with adequate fidelity to maintain its information content (6). Consequently, this type of primer elongation reaction mat have been a prebiotic route to longer RNAs.
New developments in this research include studies in the concurrent reaction of two or more activated nucleotides on montmorillonite. Significant sequence- and regio-selectivity was observed in these reactions demonstrating that a random mixture of all possible structures are not formed in the montmorillonte-catalyzed reaction. The primer-initiated montmorillonite-catalyzed synthesis of longer RNAs, with two or more different monomer units, will also be described. The reactivity in the 1-methyladenine activated (7) in template-directed synthesis and primer elangation will be discussed and new findings on the reaction pathway for the monomorillonite-catalyzed formation of RNA will be presented.
References and Footnotes
1. Gilbert, W., Nature 319, 618 (1986).
2. Ferris, J. P. and Ertem, G., J. Amer. Chem. Soc. 115, 12270-12275,
1993.
3. Ertem, G. and Ferris, J. P., J. Amer. Chem. Soc. 119, 7197-7201,
1997.
4. Ferris, J. P., Hill, A. R., Jr., Orgel, L. E., Nature 381, 59-61,
1996.
5. Szostak, J. W. and Ellington, A. D., In Gesteland, R. F. and Atkins,
J. F. (eds) The RNA World, Cold Spring Harbor Laboratory Press, Cold
Springs Harbor, New York, pp 511-533 1993.
6. Joyce, G. F., Orgel, L. E., In Gesteland, R. F. and Atkins, J. F. (eds)
The RNA World, Cold Spring Harbor Laboratory Press, Cold Springs
Harbor, New York, pp
7. Prabahar, K. J., Ferris, J. P., J. Amer. Chem. Soc. 119, 4330-4337.7,
1997.
Yury N. Vorobjev* and Jan HermansÝ
Department of Biochemistry and Biophysics,
School of Medicine,
University of North Carolina,
Chapel Hill, NC 27599-7260, USA.
*Author to whom correspondence should be addressed. Phone: (919) 966-8625;
E-mail: vorobjev@femto.med.unc.edu
ÝOn leave from the Novosibirsk Institute
of Bioorganic Chemistry,
Novosibirsk, 630090, Russia
The recently developed ES/IS method (Proteins 32, 399-413, 1998) for calculating the total conformational free energy of a solute macromolecule in water solvent is extended for the estimation of the stability of mutant proteins. The relative stability of ROP protein mutants with multiple mutations against the native protein has been estimated. The method consists of a simulation by molecular dynamics with explicit solvent molecules to produce a set of microstates of the macroscopic conformation. The implicit solvent (IS) dielectric continuum model is used to calculate the average solvation free energy as the sum of the free energies of creating the solute-size hydrophobic cavity, of the van der Waals solute-solvent interactions and of the polarization of water solvent by the soluteís charges. The reliability of the physical approximations on which the IS model is based are here tested by direct comparison with the results of microscopic simulations. The molecular surface area (the solvent excluded volume interface) interactions, when used with a microscopic surface free energy in the range 70-80 cal/(mol 2), is found to be a consistent descriptor of the hydrophobic free energy. A linear-response approximation for the water solvent reaction potential near typical polar and charged protein groups is found to be accurate to within circa 90%. A consistent set of Born radii, which reproduce the results of calculation with explicit water by the slow charging method via molecular dynamic simulation for dipeptides and solvation energies of hydrocarbons, has been determined. A faster and more reliable version of the calculation of the polarization charge density via the FAMBE method has been developed. We have earlier applied the ES/IS method to calculate the conformational free energy of native and intentionally misfolded globular conformations of proteins and have obtained good discrimination in favor of the native conformations in all instances. The estimation of mutant stability requires representation of both folded and coil states. The latter is a more complicated problem, because of greater flexibility. The study of the stability of the ROP mutants is in progress. The results of calculation on ROP mutants and a different determinants of protein stabilization will be discussed.
Genady A. Terzikian1, Ara P. Antonian1, Pogos O. Vardevanian1,
Grigir A. Manukian2 and Armen T. Karapetian2*
1Yerevan State University,
Yerevan 375049, Armenia
2Yereven Institute of Architecture and Construction,
105 Terian st. 2 bld.,
Yerevan 375009, Armenia
*Author to whom correspondence should be addressed. E-mail: ares@arm.r.am;
Fax: (3742) 56 5984.
One ofm the remarkable characteristic of the DNA double helix (ds-DNA) is the experimental fact, that the helix-coil transition of the biopolymer ocures in a highly discrete manner with many sharp steps (1). This distinctive feature is produced by the effect of the exictence of the different GC-content blocks of the base sequences in DNA. As a first approximation the block rich in AT pairs may be regarded as a low stable region in the melting process. It has been found experimentally that the melting of the separate blocks may be detected by obtaining the differential melting curve (DMC). We suggest a method of constracting the DMC by cube spline-approximation which takes acount of the instrumental error and, on the other hand it has regorous mathematical bases of existing the DMC peaks. Comparision of DMCs of "pure" DNA and its complexes with Heochst 33258 revealed the peaks corresponding to the AT rich regions of ds-DNA. Calculation showed that the GC content of these blocks is much smaller than that of the average GC-content of DNA. Revealed regions are reffered to as a premelting blocks which are responsible for early melting behavior of DNA.
References and Footnotes
1. Wada et al., CRC Grit. Rev. Biochem. 9, b7-144
(1980).
Elena V. Bichenkova1, Susan E. Austin1 Kenneth T. Douglas1*, Vladimir
N. Sil'nikov2 and Valentin V. Vlassov2
1School of Pharmacy and Pharmaceutical Sciences,
University of Manchester,
Manchester, M13 9PL, UK
2Institute of Bioorganic Chemistry,
8 Lavrent'ev Ave.,
Novosibirsk, 630090 Russia,
*Author to whom correspondence should be addressed. Phone: 44(0)161 275
2371; Fax: 44(0)161 275 2396; Email: Ken.Douglas@ man.ac.uk
To target uniquely by antisense a stretch of nucleic acid in the human genome a very long complementary oligonucleotide (15-18 base-pairs) would be required, a difficult species for drug delivery. To overcome this, the binary system of a complementary-addressing nucleic acid sequence has been proposed (1). Based on this approach, oligonucleotides conjugated to a diimidazole construct mimicking the catalytic centre of ribonuclease A have exhibited sequence-specific RNA cleavage (2).
The solution structure of the binary oligonucleotide system containing alkylimidazoles constructs was determined by high-resolution 2D NMR spectroscopy in combination with restrained molecular dynamics. The model binary system chosen, 1:2:3, comprises a 12-mer target sequence pdGTATCAGTTTCT (1), and two oligonucleotide derivatives dAGAAACp-Im (2) and Im'-pdTGATAC (3), complementary to the adjusting hexamer sequences of 1 (where Im and Im' are beta-alanylhistamine groups). The stability of complex 1:2:3 was characterized using melting experiments monitored by both UV-visible spectrophotometry and 1D NMR spectroscopy of imino protons to provide detailed structural insight into the progress of the melting of the complex. The assignment of oligonucleotide and modifying group protons was performed using 1H COSY, TOCSY and NOESY experiments. Proton-proton distance ranges calculated using the full-relaxation matrix analysis implemented in the MARGIDRAS algorithm based on the NOESY spectrum of 1:2:3, (600MHz, 200ms), were used as constraints in subsequent restrained molecular dynamics calculations. The final structure will be compared to the only other three-dimensional structural data for a binary system, incorporating pyrenyl and tetrafluoroazido groups at the interface of the short oligonucleotides (3).
References and Footnotes
1. Vlassov, V.V., Abramova ,T., Godovikova, T., Giege,
R., Silnikov, V., Sequence specific cleavage of yeast tRNAPhe with oligonucleotides
conjugated to a diimidazole construct, Antisense Nucl. Drug Devel. 1997;
7:39-42.
2. Yurchenko, L., Silnikov, V., Godovikova, T., Shishkin, G., Toulme, J.,
Vlassov, V., Cleavage of Leishmania Mini-Exon Sequence by Oligonucleotides
Conjugated to a Diimidazole Construction, Nucleosides Nucleotides 1997;
16:1721-1725.
3. Bichenkova, E.V., Marks, D.S., Lokhov, S.G., Dobrikov, M.I., Vlassov,
V.V., Douglas, K.T., Structural Studies by High-Field NMR Spectroscopy of
a Binary-Addressed Complementary Oligonucleotide System Juxtaposing Pyrene
and Perfluoro-Azide Units, J. Biomol. Struct. Dynam. 1997; 15:307-320.
Valery I. Ivanov1*, Sergei A. Bondarenko1, Evgenyi M. Zdobnov1,
Artyom D. Beniaminov1,
Elvira E. Minyat1 and Nick
B.Ulyanov2
1Engelhardt Institute of Molecular Biology,
Russian Academy of Sciences,
32 Vavilov str.,117984
Moscow, Russia
2Department of Pharmaceutical Chemistry,
University of California,
San Francisco, CA 94143-0446
*Author to whom correspondence should be addressed. E-mail: chrom@imb.imb.ac.ru
Peptidyl transferase center, PTC, is located in the large rRNA in the bigger of two ribosomal subunits; PTC is the place of protein synthesis. Here a large-scale dynamics must obviously exist, if for no other reason than the tRNA translocation from the A- to P-site. There are published data on the involvement of the protein complex L7/L12 into the translocation step of a ribosomal cycle (1). Nothing is known about the conformational switch of similar scale in the large ribosomal RNA, however. Some control mechanism could reside in PTC sending us in search of the structural prerequisites within the large rRNA.
A probable answer lies in our finding of a universal site (phylogenetically conserved), which is potentially capable of folding to pseudoknot. This site of 48 nucleotides in length comprises a portion of PTC around position 2500 (if aligned after 23S RNA of E.coli) and incorporates hairpin 89 and a very conservative sequence 5-UCGAUGU of the so called peptidyl transferase ring. So we called the found pseudoknot by term Peptidyl Transferase Pseudoknot, or PTP.
Especially interesting is the existence of the base pairs in the hairpin 89 conflicting with the PTP formation. We hypothesize that a reversible process of (pseudo)knotting-unknotting PTP at the expense of GTP hydrolysis is the aforementioned switch responsible for passing particular steps of ribosome functioning (translocation?) (2).
References and Footnotes
1. Burma, D.P., Srivastava, S., Srivastava, A.K., Mahanti.
S., Dash, D. (1986) in: Structure, Function, and Genetics of Ribosomes
(eds. S B. Hardesty, G. Kramer), Springer-Verlag, pp. 438-453.
2. Ivanov, V.I., Bondarenko, S.A., Zdobnov, E.M., Beniaminov, A.D., minyat,
E.E., Ulyanov, N.B., FEBS Letters (1999), accepted.
Supported by Russian Fund for Basic Research, Grant 95-04-11707, and State Support for Leading Scientific Schools, Grant 96-15-98093
David R. Pawlowski, Mihai Ciubotaru and Gerald B. Koudelka
Department of Biological Sciences,
State University of New York at Buffalo,
Buffalo, NY 14260
In complexes of the bacteriophage 434 binding sites with 434 repressor, the central four base pairs of the 14 base pair site are not contacted by the protein, although changes in these bases alter the binding site's affinity for repressor. Our previous data suggested that the ability of the noncontacted central bases to be overtwisted in repressor-DNA complexes governs the affinity of the binding site for 434 repressor. We know that 434 repressor prefers operators bearing A/T bases at their centers over ones bearing G/C base pairs at their central sequences. One model to explain these observations suggests that the resistance of H-bonds between bases in the twisted regions to be distorted modulates the twisting flexibility DNA. This idea was tested by examining the affinity of 434 repressor synthetic DNA binding sites that bear non-natural bases at one or more of the central four positions. Consistent with the model, repressor prefers to bind operators bearing nebularine-cytosine base pairs, a base pair containing only one base pair H-bond, over those bearing guanine-cytosine base pairs, a base pair with three base pair H-bonds, at their centers. Similarly, repressor binds more tightly to operators bearing A-T bases, which have two base pair H-bonds, at their centers, than it does to a binding site bearing 2-aminopurine-thymine base pairs which contain three base pair H-bonds. Together, these data suggest that the number of base pair hydrogen bonds between bases at the center of the 434 binding site, in part, governs its affinity for 434 repressor.
In vitro selection experiments were conducted in an effort to uncover the sequence determinants that govern DNA twist and/or twisting flexibility. Using a ring closure assay, we selected DNA sequences from a randomized pool of sequences that enhance either the formation of overwound and underwound DNA topoisomers. Sequence analysis of individual selected DNAs indicated that the absence or presence of a 5íC-A3í base step is the dominant indicator of the overall twist of the DNA, with nearly each DNA sequence found in the overwound topoisomer pool bearing at least one C-A step. Detailed sequence analyses of the individual clones are completely consistent with the dependence of twist on sequence detailed by Gorin et al. (1995). Surprisingly, the population of sequences isolated from the overwound pool of topoisomers bound repressor more poorly than did those isolated from the underwound pool. This observation highlights the importance of DNA twisting flexibility in governing the strength of 434 repressor-DNA interactions.
References and Footnotes
1. Gorin, A.A., Zhurkin, V.B., Olson, W.K., (1995) B-DNA Twisting Correlates with Base-pair Morphology, J. Mol. Biol. 247, 24-48.
Karolin Luger1, Armin W. Muder2, David F. Sargent3 and
Timothy J. Richmond3
1Department of Biochemistry and Molecular Biology,
316 Molecular and Radiological Biosciences Building,
Colorado State University,
Fort Collins, CO 80523
2UBS,
CH-8000 Zürich, Switzerland
3Institute for Molecular Biology and Biophysics|
ETH Honggerberg,
CH-8093 Zürich, Switzerland
*Author to whom correspondence should be addressed. Phone: (970) 491-6405;
Fax: (970) 491-0494; E-mail: <kluger@lamar.colostate.edu>
The nucleosome core particle, the basic repeating unit of chromatin in all eukaryotic cells, consists of an octameric protein core around which 147 base pairs of DNA are wrapped in 1.65 superhelical turns. The protein core itself is composed of two copies each of the four histone proteins H2A, H2B, H3, and H4. The histone proteins contain a common conserved structural motif, the histone fold, whose rigid framework is responsible for the binding of most of the DNA within the protein core, and the flexible histone tails, which are implicated in inter-particle contacts. The compaction of nucleosomal DNA by a factor of five is the result of the histone-induced bending and close packing of the DNA. The repeating nucleosome cores further assemble into higher order structures that are stabilized by the histone tails and by the linker histone H1.
We present the 2.0 resolution structure of the nucleosome core particle containing a defined sequence DNA fragment. The structure of the 206 kDa protein-DNA complex reveals the form of DNA that is predominant in the living cell. Recent studies imply that chromatin is highly dynamic, allowing transcription and replication of the DNA while maintaining a high degree of compaction. This propensity for unfolding and refolding stems from the structural design of the nucleosome core, in which the DNA is held by the histone octamer at fourteen independent attachment points. The strength of protein-DNA interaction is strong at the center and relatively weak at the entry- and exit points of the DNA to facilitate dissociation of protein and DNA during replication and transcription of nucleosomal DNA.
The 2.0 data allow us to locate numerous solvent molecules, many of which
appear to play an instrumental role in shaping the DNA into a tight supercoil.
In addition, ordered water molecules between the structural entities of
the histone octamer might facilitate partial dissociation of the histone
dimers from the histone tetramers due to entropic effects.
Nicola G. A. Abrescia, Lucy Malinina and Juan A. Subirana
Departament d'Enginyeria Química,
Universitat Politècnica de Catalunya,
Diagonal 647, Barcelona E-08028, Spain
We have determined the structure of the alternating (YR) decanucleotide d(CGTATATACG) by single crystal X-ray diffraction in two different space groups. A high resolution structure (1.58 ) was obtained by complexing with the netropsin drug. Both structures were solved by molecular replacement and refined with either the X-Plor or the Shelx programmes.
The general conformation is B-like with an end-to-end interaction which involves terminal guanines as described by Spink et al. (PNAS 92, 10767, 1995). One crystal belongs to space group P41212 with a=b=53.46, c=101.49 . This is a new way of packing for decamers. A novel C·A·T triplet structure has also been tentatively identified. In the high resolution structure the overall conformation is B-like but the end-to-end interaction previously seen is different. In fact one of the terminal guanines stacks on a pyrrole ring of netropsin in the minor groove of the helix and the terminal cytosines are completely disordered. The crystal belongs to space group P212121 with a=25.18, b=39.11, c=53.47 .
Both structures are characterized by the specific association with nickel ions, involving the N7 atom of every guanine. In the structure at 1.58 each metal ion has a complete hydration shell with an octahedral coordination geometry. Unequivocally all water molecules around nickel ions have been determined. Also one Ni2+ ion in P212121 is directly coordinated with the O2P of adenine of a symmetry related molecule while in the P41212 structure it is shared between two symmetry related guanines. Until now no oligonucleotide has been crystallized in the presence of Ni2+. Thus it appears that such ions may be used as specific probes for guanine in nucleic acids.
J. Sponer
J. Heyrovsky Institute of Physical Chemistry,
Academy of Sciences of the Czech Republic,
Dolejskova 3,
182 23 Prague, Czech Republic
Interactions of nucleic acid bases (H-bonding, stacking, interactions with cations) significantly infuence structure and dynamics of nucleic acids. Recent advances of computational chemistry (high level ab initio quantum-chemical calculations with inclusion of electron correlation effects and classical nanosecond molecular dynamics simulations of hydrated nucleic acids) resulted into a major improvement of the understanding of the role of base - base interactions in nucleic acids.
Recent ab initio calculations provided the ultimate picture of the physical origin of base stacking and base pairing interactions. Further, the quantum chemical data provide a solid basis for parametrization and verification of molecular-mechanical force fields and substantially reduce the uncertainty concerning the accuracy of force fields.
Ab initio calculations are still essential in studies of interactions which are not properly approximated by pair-additive molecular mechanical force fields. The interactions involving metal cations represent a particularly important area where quantum chemistry can hardly be challenged by other theoretical techniques.
Nanosecond-length molecular dynamics simulations represent a robust tool (after the potential is verified) to study the actual role of the base - base interactions in nucleic acids. The picture obtained by ab initio calculations and MD simulations can differ substantially, and some examples will be given. The major advantage of MD simulations over the ab initio treatment is the explicit inclusion of solvent and consideration of sufficiently large fragments of molecules.
J. Sponer1, J.V. Burda2,
J.E. Sponer1, M. Sabat3,
B. Lippert4, P. Hobza1
and J. Leszczynski5
1J. Heyrovsky Institute of Physical Chemistry,
Academy of Sciences of the Czech Republic,
Dolejskova 3,
182 23 Prague, Czech Republic
2Department of Chemical Physics,
Faculty of Mathematics and Physics,
Charles University,
Prague, Czech Republic
3Department of Chemistry,
University of Virginia,
Charlottesville, Virginia 22901
4Department of Chemistry,
University of Dortmund,
44221 Dortmund, Germany
5Department of Chemistry,
Jackson State University,
Jackson, Mississippi 39217
Recent developments of computer hardware and software allowed extensive ab initio investigation of interactions of metal cations with fragments of nucleic acids. These calculations provide an advanced physico-chemical description of the metal-cation complexes and their energetics with a reliable inclusion of all contributions including polarization effects and charge transfer. In the present contribution we discuss four topics where the recent ab initio calculations illuminated several new important aspects of cation - nucleobase interactions.
i) Specific differences among cations interacting with nucleic acid bases,
mainly the difference between zinc and magnesium.
ii) Stabilization of Watson Crick and mismatch base pairs by polarization
effects due to cation binding to the N7 position of purine bases.
iii) Interactions of metal cations with nucleotides.
iv) Metal-cation stabilized rare tautomers and mispairs of bases.
Special attention will be paid to differences between ab initio calculations carried out in gas phase, and the condensed phase and solid state experiments.
Systems studied in gas phase are often dominated by electrostatic effects due to the charge residing on the metal entity. On the other hand in experiments the electrostatic effects are often completely suppressed by solvent screening and counterions. This frequently (but not always) leads to controversies between the ab initio predictions and the experimental observations. The major point is that the degree of neutralization of the electrostatic effects differ from one experimental arrangement to another. In DNA, the expression of cation charge will depend on the particular DNA architecture, DNA sequence and its environment. Quantum chemical theory and experiment show the metal cation - nucleobase complexes under quite different conditions. The ab initio theory and experiments (condensed phase, x-ray) complement each other and both are important to properly understand the role of cation binding to bases in DNA.
References and Footnotes
1. J. Sponer, J.V. Burda, M. Sabat, J. Leszczynski, P. Hobza, J. Phys. Chem. A 102, 5951-5957 (1998) and references therein.
N. Spaèková1, I. Berger2 and J. Sponer3
1Institute of Biophysics,
Academy of Sciences of the Czech Republic,
Královopolsk· 135, 612 65 Brno, Czech Republic
2Institute for Molecular Biology and Biophysics,
ETH-Hönggerberg, CH-8093 Zurich, Switzerland.
3J. Heyrovsky Institute of Physical Chemistry,
Academy of Sciences of the Czech Republic,
Dolejskova 3, 182 23 Prague 8, Czech Republic
We have carried out a set of nanosecond-scale molecular dynamics simulations of various unusual nucleic acid structures: four-stranded i-DNA molecules, and four-stranded parallel and antiparallel G-DNA molecules. Total length of all simulations exceeded 60 ns and the study thus belongs to the most extensive MD projects carried out on nucleic acids so far. Starting structures have been taken from high-resolution oligonucleotide crystal data and NMR studies. The calculations provided a new insight into the role of base stacking interactions in these molecules. Also the behaviour of loop regions of G-DNA molecules and the influence of cations on their stabilization has been characterized. In most cases an excellent agreement has been found between theoretical and experimental structures. All simulations were carried out using the AMBER95 force field and the particle mesh Ewald method for long-range interactions. The simulations were supplemented with high-level ab initio calculations to estimate the accuracy of the force field and to evaluate electronic effects not included in the pair-additive force field.
References and Footnotes
1. N. Spaèková, I. Berger, M. Egli and J.
Sponer, J. Am. Chem. Soc. 120, 6147 (1998).
2. N. Spaèková, I. Berger and J. Sponer, J. Am. Chem. Soc.
in revision (1999).
3. J. Sponer, H.A. Gabb, J. Leszczynski and P. Hobza, Biophys. J. 73,
76 (1997).